TRPA1 and TRPV4 inhibitors and methods of using the same for organ-specific inflammation and itch

ABSTRACT

Provided are methods of treating and/or preventing dermatological disorders. Provided are methods of reducing skin inflammation, reducing pain, and/or reducing itch in a subject in need thereof. The methods may include administering to the subject an effective amount of a TRPA1 and/or TRPV4 inhibitor. Further provided are compositions including a TRPA1 and/or TRPV4 inhibitor compound in combination with a carrier, vehicle, or diluent that is suitable for topical application.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/101,883, filed Jan. 9, 2015, which is incorporated herein byreference in its entirety.

SEQUENCE LISTING

The sequence listing is filed with the application in electronic formatonly and is incorporated by reference herein. The sequence listing textfile “028193-9128-US05_As_Filed_Sequence_List.txt” was created on Mar.22, 2016, and is 698 bytes in size.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersDE018549 and DE018529S1 awarded by the National Institutes ofHealth/National Institute of Dental and Craniofacial Research(NIH/NIDCR), and grant numbers AR059402, AR31737, and AR050452 awardedby the National Institutes of Health/National Institute of Arthritis andMusculoskeletal and Skin Diseases (NIH/NIAMS). The government hascertain rights in the invention.

FIELD

This disclosure relates to methods and compositions for treatinginflammation, pain, itch, cancer, autoimmune diseases, fibroticdiseases, skin pigmentation, and/or other dermatological disorders.

INTRODUCTION

The skin is the largest organ in many vertebrates, including humans. Itprovides barrier protection against the potentially harmful externalenvironment. The skin also represents the site of first interaction ofthe ambient environment to immunologically competent and sentientstructures of the organism. Cells endowed with sensory transductioncapacity for warmth, cold, mechanical cues, pain, and itch are sensoryneurons in the dorsal root and trigeminal ganglia with their peripheralaxons directly interfacing with skin. However, successfully targetingthe skin for treatment of inflammation, pain, itch, cancer, autoimmunediseases, fibrotic diseases, skin pigmentation, and other dermatologicaldisorders has remained elusive.

Biochemical pathways in to the skin include those relating to thetransient receptor potential (TRP) superfamily of ion channels. One ionchannel in this family is TRPV4. TRPV4 is a multimodally-activatednon-selective cation channel permeable to calcium (i.e., Ca++). Inepidermal keratinocytes of mammalian skin, the TRPV4 ion channel isexpressed robustly. However, TRPV4 is also expressed in skin-innervatingsensory neurons. In Trpv4−/− mice, an epidermal phenotype of impairedbarrier function between epidermis and dermis has been shown. In regardsto pain signaling, TRPV4 has been found critical for physiologicalwithdrawal responses to noxious osmotic and mechanical, but not thermalcues, and has also been found relevant for inflammation ornerve-damage-induced sensitization of nociception. While it isunderstood that TRPV4 is expressed in epidermal keratinocytes andskin-innervating sensory neurons, an in vivo role of TRPV4 inpathological pain evoked by UVB exposure has not been demonstrated.Moreover, a direct role of TRPV4 in itch transmission has not beendemonstrated as of yet. TRPA1 is another TRP ion channel located on theplasma membrane. TRPA1 acts as sensor for environmental irritants, pain,cold, and stretch. Although TRPV4 and TRPA1 function in the skin, it isnot known whether targeting TRPV4 and/or TRPA1 would be useful in thetreatment of inflammation, pain, itch, cancer, autoimmune diseases,fibrotic diseases, skin pigmentation, and other dermatologicaldisorders. Furthermore, specific TRPV4 and TRPA1 inhibitors are notpresently known. New and successful treatments for dermatologicaldisorders are needed.

SUMMARY

In an aspect, the disclosure relates to methods of treating and/orpreventing a fibrotic disease in a subject in need thereof. The methodsmay include administering to the subject an effective amount of a TRPV4and/or TRPA1 inhibitor. The fibrotic disease may be selected frompathologic skin fibrotic response, synovial fibrosis, and lung fibrosis.The pathologic skin fibrotic response may be selected from skinfibrosis, keloid formation, chemical or actinic injury, a pathologicresponse to skin irradiation (“radio-derm”), psoriasis, chronic atopicdermatitis, chronic UV-dermatitis, scleroderma, and conditionscharacterized by a chronic maladaptive injury response to an ongoingnoxious insult to the skin or inappropriate scar formation. The TRPV4and/or TRPA1 inhibitor may be a compound according to Formula I:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups; D is C₁-C₃ alkylene; E is a bond, or C₁-C₂ alkylene; and R isselected from the group consisting of hydrogen, hydroxyl, amino, alkyl,alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring. The TRPV4and/or TRPA1 inhibitor may be at least one compound selected from thefollowing:

In a further aspect, the disclosure relates to methods of treatingand/or preventing a dermatological disorder in a subject in needthereof. The methods may include administering to the subject aneffective amount of a TRPV4 and/or TRPA1 inhibitor. The dermatologicaldisorder may be selected from inflammation, pain, itch, cancer,autoimmune diseases, fibrotic diseases, skin pigmentation, and/or otherdermatological disorders. The TRPV4 and/or TRPA1 inhibitor may be acompound according to Formula I:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups; D is C₁-C₃ alkylene; E is a bond, or C₁-C₂ alkylene; and R isselected from the group consisting of hydrogen, hydroxyl, amino, alkyl,alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring. The TRPV4and/or TRPA1 inhibitor may be at least one compound selected from thefollowing:

In an aspect, the disclosure relates to methods of reducing skininflammation in a subject in need thereof. The methods may includeadministering to the subject an effective amount of a TRPV4 and/or TRPA1inhibitor. The skin inflammation may be related to UVB exposure. Theskin inflammation may be associated with a dermatological disorderselected from sunburn, rosacea, Xeroderma pigmentosum, non-melanoma skincancer, and photoaging, or with a disorder selected from non-UV skinburn, disturbed wound healing, and pain of bone fractures. The methodmay further include reducing pain in the subject. The TRPV4 and/or TRPA1inhibitor may be a compound according to Formula I:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups; D is C₁-C₃ alkylene; E is a bond, or C₁-C₂ alkylene; and R isselected from the group consisting of hydrogen, hydroxyl, amino, alkyl,alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring. The TRPV4and/or TRPA1 inhibitor may be at least one compound selected from thefollowing:

In a further aspect, the disclosure relates to methods of painmanagement. The methods may include administering to at least a portionof the skin of a subject in need thereof an effective amount of a TRPV4and/or TRPA1 inhibitor. The pain may be associated with a dermatologicaldisorder selected from sunburn, rosacea, Xeroderma pigmentosum,non-melanoma skin cancer, and photoaging, or with a disorder selectedfrom non-UV skin burn, disturbed wound healing, and pain of bonefractures. The method may further include reducing pain in the subject.The TRPV4 and/or TRPA1 inhibitor may be a compound according to FormulaI:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups; D is C₁-C₃ alkylene; E is a bond, or C₁-C₂ alkylene; and R isselected from the group consisting of hydrogen, hydroxyl, amino, alkyl,alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring. The TRPV4and/or TRPA1 inhibitor may be at least one compound selected from thefollowing:

Another aspect of the disclosure provides methods of reducing itch in asubject in need thereof. The methods may include administering to thesubject an effective amount of a TRPV4 and/or TRPA1 inhibitor.

In a further aspect, the disclosure relates to compositions including aTRPV4 and/or TRPA1 inhibitor compound in combination with a carrier,vehicle, or diluent that is suitable for topical application.

In a further aspect, the disclosure relates to topical formulationsincluding a TRPV4 and/or TRPA1 inhibitor, wherein the TRPV4 and/or TRPA1inhibitor may be a compound according to Formula I:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups; D is C₁-C₃ alkylene; E is a bond, or C₁-C₂ alkylene; and R isselected from the group consisting of hydrogen, hydroxyl, amino, alkyl,alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring. The TRPV4and/or TRPA1 inhibitor may be at least one compound selected from thefollowing:

In a further aspect, the disclosure relates to novel TRPV4 and/or TRPA1inhibitors. The TRPV4 and/or TRPA1 inhibitor may be at least onecompound selected from the following:

The inhibitors may inhibit TRPV4. The inhibitors may inhibit TRPA1. Theinhibitors may inhibit TRPV4 and TRPA1. The TRPA1 inhibitors may notinhibit TRPV1, TRPV2, or TRPV3. The inhibitor may be specific for TRPV4.The inhibitor may be specific for TRPA1. The inhibitor may be specificfor TRPV4 and TRPA1.

The disclosure provides for other aspects and embodiments that will beapparent in light of the following detailed description and accompanyingFigures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Keratinocyte-specific and inducible Trpv4 null mouse and its UVBresponse. (A) Gene-targeting of Trpv4 and genetic manipulationunderlying generation of keratinocyte-specific and inducible Trpv4knockout mice. Shown are sequential steps of mouse Trpv4 targeting,starting with flanking Trvp4 exon13 with loxP elements and insertion ofa selection cassette, flanked by frt sites, in mouse embryonic stemcells. After generation of chimeric mice and stable transmission of theengineered mutation, the selection cassette was removed by breeding toFLPe mice. Resulting mice were homozygosed and crossed withK14-CRE-ER^(tam) mice, which then permitted keratinocyte-specific andinducible Trpv4 knockout/knockdown. (B) DNA genotyping. Shown are PCRproducts of WT, heterozygote and homozygous Trpv4^(lox/lox) mice. Notethat the PCR products needed to be digested with PacI, and that all micewere pre-screened to be CRE+ by another genotyping PCR. (C) Co-labelingof mouse skin for keratin-1 and keratin-14 indicate the establishedpattern for vehicle-induced control mice (upper panel), and a similarpattern for specific TRPV4 knockdown in keratinocytes (lower panel).However, in these animals note a slightly increased expression of K14 inthe stratum spinosum, reflecting attenuated TRPV4 expression and thusreduced Ca⁺⁺ influx. K14 is normally down-regulated at thebasal-to-suprabasal transition, concomitant with the rise inCa⁺⁺-signaling and induction of terminal differentiation. (D) TRPV4protein expression in L5 DRG neurons not different between genotypes.Densitometry of TRPV4 immunohistochemistry in L5 (=foot-pad innervating)DRG neurons (upper panel micrographs), the bar-diagram illustrates thelack of a difference in terms of TRPV4 protein abundance in oil- vs.tam-treated mice, for both base-line and 48 hours after UV exposure,confirming the specificity of TRPV4 knockdown in skin when using K14 asCRE driver. Note the characteristic morphology of decorated cellsidentifying them as DRG sensory neurons. Note also the different levelsof TRPV4 expression in these neurons, as noted previously; n=3mice/group, ≥50 neurons/mouse. (E) Lack of TRPV4 expression in Merkelcells in foot-pad epidermis. A confocal triple-fluorescent micrographpanel is shown, depicting representative images of immuno-labeledpaw-pads from iKO control vs. tamoxifen-induced mice. Note completeknockdown of TRPV4 in this example (red channel). For Merkel cells(green channel), an anti-cytokeratin 8 antibody was used. Note lack ofTRPV4 co-labeling in Merkel cells. Blue channel=DAPI. (F) Lack of effectof tamoxifen application in K14-CRE-ER^(tam) mice on UVB behavioralsensitization. Note very similar withdrawal thresholds in(K14-CRE-ER^(tam) X Trpv4^(lox/+)) mice (=Trpv4 heterozygotes inkeratinocytes when induced with tamoxifen) for noxious mechanical (upperdiagram) and thermal (lower diagram) stimulation; n=7 mice per group.Also note the time-course with peak sensitivity at time-point 48 hours.(G) Size distribution of pERK-expressing L5 DRG neurons in oil-treatediKO mice, exposed to UVB. The bar diagram illustrates size prevalence ofsmall and medium-size sensory neurons that express pERK 48 hours afterUVB exposure, note absence of larger neurons (>1200 μm²), n=22 neurons.

FIG. 2: UVB stimulation device and UVB keratinocyte control experiments.(A) UV spectrum emitted by the LEDs, overlapped with the spectrum ofquartz (red trace), which is almost fully permeable to UVB, and glass(blue trace), which has a very low UVB permeability. (B) Focusingproperties of the ball lens. (C) Focal geometry of the combination ofUV-LED and ball lens. (D) Absence of thermal effects of the UV-LEDs;measurement of temperature in the focal point over time. (E) TRPV3activation experiment. Induction of a Ca++ transient by camphor, whichcan be blocked effectively by 10 μM IPP, suggesting TRPV3-mediatedsignaling. (F) TRPV4 selective activator GSK101-related findings. Ca++transient in 1° MK in response to 5 nM GSK101, which can be completelyblocked by 20 μM GSK205, suggesting it is specifically mediated byTRPV4. The GSK101-response can also be eliminated by absence of externalCa++, in keeping with TRPV4 signaling. (G) TRPV4 is sufficient for theUVB-Ca++ response—HEK293T cell heterologous transfection. Directedexpression of TRPV4 in HEK293T cells leads to a Ca++-transient inresponse to UVB radiation, which is greatly reduced incontrol-transfected cells. Preexposure to 20 μM GSK205 virtuallyeliminates the Ca++-signal in TRPV4-transfected cells, and eliminatesthe moderate signal of control-transfected cells.

FIG. 3: Keratinocyte-specific ablation of Trpv4 leads to alterations innocifensive behavior in response to UVB. (A) Epidermal TRPV4 expressionand its loss upon keratinocyte-specific ablation of Trpv4 in tam-inducediKO mice. (i) TRPV4 immunofluorescence. Note TRPV4 in epidermis ofvehicle (oil) treated control, but not tam-induced iKO mice. Bar=10 μm.(ii) Western blot of epidermal lysates from paw-pad skin. Note knockdownand more complete loss of TRPV4 following induced Trpv4-ablation(β-actin used for normalization). (iii) qRT-PCR for Trpv4 mRNA frompaw-pad skin is shown, indicating significant Trpv4 knockdown inresponse to tam-treatment vs. carrier (oil). P<0.0001, t-test. (iv)Immunofluorescence for epidermal lineage markers. In WT skin, basalepidermal marker keratin-14 is downregulated and suprabasal markerkeratin-1 is induced upon commitment to terminal differentiation. Uponknockdown of TRPV4, this balance appears perturbed, with some spinouslayer cells showing co-labeling. Bar=10 μm. (B) Nocifensive behavior inresponse to UVB exposure. Time-course (in hours) for nocifensivebehavior elicited by either a noxious mechanical stimulus (automatic vonFrey hair assay, left) or thermally-evoked nocifensive behavior(Hargreaves' assay, right). Note significantly less sensitization inTrpv4^(−/−) and in tam-treated iKO mice, relative to oil-treated(vehicle) iKO and WT mice. n≥10 animals per group; ** p<0.01 ANOVA. (C)Correlation between nocifensive behavior and level of Trpv4 knockdown.n=12 animals are shown for which both parameters were available andTrpv4 mRNA levels <0.45. Note the four vehicle-induced animals (greensymbols) vs. their tamoxifen-induced counterparts (red symbols). (D)Loss of epidermal TRPV4 shows no significant effect on nocifensivebehaviors caused by formalin injection. Bars depict average cumulativenocifensive behavior within the first 10 minutes (phase I), and 10-45minutes (phase II) post-injection. n=4 per group. (E) Phosphorylated ERKin L5 DRG neurons. pERK immunofluorescence of L5 DRG sections are shownfor oil- and tam-treated iKO animals±exposure to UVB. Note that onlyUVB-exposed control mice show pERK expression in the paw-pad-innervatingL5 DRG. Quantifications are shown at right. n=3 animals per group, 6sections per DRG per animal, ** p<0.01 ANOVA.

FIG. 4: Structural and ultrastructural analyses showing thatUVB-mediated skin tissue injury depends upon keratinocyte TRPV4, andImmuno-histochemical analysis demonstrates that UVB-mediated activationof keratinocytes and recruitment of macrophages and neutrophils dependsupon keratinocyte TRPV4. (A) 1 μm toluidine-blue semi-thin sections.Micrographs show representative findings of skin in response to UVB,sampled 48 hours after UVB exposure. Note that upon UVB stimulation,oil- (TRPV+) but not tam-treated (TRPV−) iKO mice exhibit separations atthe epidermal-dermal boundary and robust signs of tissue injury; notegranulocytes (Gr, neutrophil). Note also that just beneath the stratumcorneum (SC), the upper epidermis shows extensive structural damagewhich could also be seen in skin of tam-treated iKO mice where Trpv4knockdown was incomplete, but not in those animals where it was morecomplete (see FIG. S2A). Bars=20 μm. Der=dermis; Epi=epidermis. (B)Ultrastructural findings by EM. Selected areas from 1 μm semithinsections of paw skin were examined by transmission electron microscopy.(A-A′) and (C-C′) show normal epidermal (Epi) structure for both, oil-and tam-treated iKO mice, in the absence of UVB stimulation. (A) and (C)show and intact epidermis. Basal (BL) and spinous (Sp) layers aremagnified A′ and C′ displaying a normal organization with no evidence ofepidermal damage. (B,B′B″), (D-D′) and (E-E′) show representativefindings of skin in response to UVB, sampled 48 h after UVB exposure.(B) Disrupted epidermis in oil treated iKO mice. An area equivalent tothe boxed area is magnified in (B′), where granulocyte infiltration ofepidermis is evident (Gr) and blistering with detachment of theepidermis from the dermis (double arrows). (B″) Upper part of epidermisin contact with stratum corneum (SC), showing extensive vacuolizationand deposits of fibrin inside the vacuoles (asterisks). (D) Tamoxifentreated iKO mice with incomplete knockdown of trvp4 show similar skinphenotype to oil treated iKO mice, with robust signs of tissue damage tobasal and spinous layer, fibrin deposits (asterisks) and intercellularspaces (arrowheads in D′). (E) Intact epidermis in iKO with completeknockdown of trvp4, with normal basal and spinous layers in (E′). Der,dermis. Dotted lines indicate the dermo-epidermal boundary. Bars=20 μmfor A, B, C and D; 10 μm for B′ and E′ and 2 μm for the othermicrographs. (C) IL-6 upregulation in keratinocytes as marker ofepidermal activation. IL-6 immunofluorescence reveals a reduced abilityof TRPV4-deficient mice to elevate keratinocyte IL-6 expression inresponse to UVB exposure. Quantifications for protein is shown next tomicrograph. Densitometries are for n≥3 mice per group, showingsignificant upregulation for oil-treated iKO mice, lack thereof fortam-treated. Right-hand bar diagram shows 11-6 mRNA quantification andtime-course. II-6 mRNA was determined by qPCR after isolation of totalRNA from paw-pad epidermis. Note the early and robust increase, albeitwith variation, at the 2 hour time-point, in WT control epidermis, incontrast the very moderate increase in Trpv4^(−/−) epidermis. Note alsothe sustained robust upregulation at 24 hours, again moderatelyupregulated in Trpv4^(−/−) epidermis. Quantifications are for n=8-12mice/group. * denotes statistically significant (p=0.011, t-test);scale-bar=20 μm. (D) Recruitment of macrophages in UVB-exposed skin.Note that the numbers of dermal CD68+ macrophages induced byUVB-exposure in control mice is significantly reduced when Trpv4 isablated in the epidermis. Quantifications are shown at right (n=3mice/group; * p<0.05 t-test); scale-bar=20 μm. (E) Recruitment ofelastase-expressing neutrophils to UVB-exposed skin. Shown arerepresentative immunofluorescence micrographs and respectivequantifications. Note a strong increase in abundance ofelastase-expressing neutrophils in control mice, and a lack thereof intam-treated iKO mice. (n=4 mice/group, * p<0.05 t-test); scale-bar=40μm.

FIG. 5: Histopathology in Trpv4−/− and control mice in response to UVB.(A) Trpv4 knockdown level of samples shown in FIG. 4A-E. This bardiagram shows relative level of knockdown of Trpv4 in comparison withWT, of UVB-exposed skin samples shown in FIG. 4. An adjacent sample ofhindpaw skin was RNA-extracted at 48 h post-exposure and subjected toTrpv4 qRT-PCR; pooled WT mRNA values from 10 mice were set as 100%. (B)Light microscopic analyses of 1 μm semithin sections findings fromTrpv4−/− and WT control mice. Normal skin is shown in the upper row forboth genotypes in the unstimulated state, presence of epidermal anddermal inflammation in WT control vs. absence thereof in Trpv4−/− whenexposing the skin to UVB, sampling conducted at 48 hours. Noteinflammatory changes similar to those of oil-treated iKO mice, as shownin FIG. 4. (C) Ultrastructural analyses of Trpv4−/− and WT control mice.(A-A′) and (B-B′) WT and Trpv4−/− mice show normal skin morphology withintact epidermis (Epi) in the absence of UVB stimulation. A′ and B′ showhigher magnification of basal layer (BL) cells. (C-C′) Damaged epidermiswith vacuolization (inset in C) and granulocyte (neutrophil)(granulocyte—Gr) infiltrate (C′). (D-D′) Normal epidermal and dermalultrastructure in Trpv4−/− mice exposed to UVB. Der—dermis; NT, nerveterminals. Dotted lines indicate the dermo-epidermal boundary. Bars=10μm for A, B, C, C′ and D and 2 μm for the other micrographs. (D) IL-6upregulation in epidermal keratinocytes in response to UVB depends onTrpv4; findings from Trpv4−/− and WT control mice. Fluorescentmicrographs from Trpv4−/− and WT control skin, unexposed and exposed toUVB are shown. Note strong IL-6 signal in WT, exposed to UVB, and lowsignal in Trpv4−/− for both non-exposed and UVB-exposed states. Alsonote IL-6-expressing innervating peripheral nerve endings in the dermis.(E) No difference in mast cell abundance in UVB-photodermatitis in iKOmice. Left-hand micrograph shows mast-cells within sub-epidermalinflammatory tissue, stained with toluidine-blue, in an iKO mouseinduced with tamoxifen, right micrograph its counterpart in anoil-treated iKO mouse. Mast-cells are indicated by white arrow-heads.Bar=20 μm. Right-hand bar diagram indicates quantification of mast-cellcount per 63× visual field (5 fields per mouse, 3 mice per group).

FIG. 6: Ca++ influx into keratinocytes in response to UVB depends onTRPV4. (A) Custom-built UVB cell illumination apparatus. See also FIG.2. (B) Fluo-4 Ca++ imaging in 1° MKs. Fluorescent micrographs of 1° MKsafter loading with Ca++-sensitive dye, fluo-4, before (upper) and at theend of UVB exposure (lower). Bar=10 μm. C-H UVB-evoked Ca++ signalingprofiles. Fluo-4 imaging was used to detect Ca++ transients in 1° MKsfollowing UVB exposure. y-axis indicates the increase in fluorescence,ΔF, normalized for prestimulation signal, F0 (ΔF/F0). The signal shownis that averaged from ≥50 cells. (C) Ca++ signaling is dependent uponUVB, and is strikingly reduced when quartz coverslips are replaced byglass ones, which prevent UVB permeation (see FIG. 2A). Note that thisparticular Ca++ signal in WT 1° MKs persisted after UVB, as is sometimesobserved. (D) UVB-evoked Ca++ signaling is dependent on external [Ca++].(E) UVB-evoked Ca++ signaling is not seen in Trpv4−/− 1° MKs, revealingthe importance of the TRPV4 ion channel. (F) UVB-evoked Ca++ signalingis strongly down-regulated in the presence of TRPV4-selective inhibitor,GSK205 (20 μM). (G) The UVB-evoked Ca++ signal is not inhibited by theTRPV3-selective inhibitor, IPP. For validation of IPP's activity, seeFIG. 2E. (H) The UVB-evoked Ca++ signal can be strongly reduced withspecific PLC inhibitor, U73122.

FIG. 7: Central role for keratinocyte TRPV4 in UVB-evoked Ca++ signalingand nocifensive behavior—effects of ET1. (A) Effects of ET1 onUVB-evoked Ca++ signaling in 1° MKs. Panel (i) shows averaged Ca++transients in 1° MK in response to UVB, their augmentation byco-exposure to ET1 peptide, and their significant attenuation by eitherGSK205, which inhibits TRPV4, or ET-convertase inhibitor CGS35066, whichblocks ET1 proteolytic processing. Panel (ii) shows ET-augmented,UVB-induced Ca++ transients as in (i), but in this case, where they areattenuated by selective antagonism of ET(R)-A (BQ123) and ET(R)-B(BQ788). Panel (iii) illustrates the complete elimination of theET1-augmented Ca++ transients when both subtypes of ET(R) are blocked.(B) 4α-PDD-evoked Ca++ signaling in 1° MKs—ET1-related findings.Left-hand panel shows Ca++ transients (as per fura-2 ratiometricimaging) in response to the selective TRPV4 activator 4α-PDD. Asignificant increase in the response can be observed by co-applicationof ET1, and this is partially dependent on ET(R)-A and completelydependent on ET(R)-B. (C) Upregulation of ET1 in mouse paw in responseto UVB. Immunohistochemistry reveals a significantly stronger ET1 signalin UVB-exposed skin of oil-vehicle-treated (TRPV4+) rather thantamtreated iKO mice. Quantifications are for n=3 mice/group. *** denotesstatistically significant (p<0.001, t-test). (D) Nocifensive behavior inresponse to ET1 footpad injection depends on epidermal TRPV4. Bardiagram summarizes behavioral findings for Trpv4−/−vs. WT and foroil-treated vs. tam-treated iKO mice. Note that in WT and oil-treatediKO mice, footpad injection of ET1 leads to significant levels ofmechanical allodynia. Trpv4−/− and tam-treated iKO mice fail to respond;n≥7 mice/group, ** p<0.01, ANOVA.

FIG. 8: Central role for KC TRPV4 in UVB-evoked Ca⁺⁺ signaling andnocifensive behavior—ET1-related supplementary findings. (A)Augmentation of GSK101-evoked Ca⁺⁺ signaling by ET1. Shown are averagedCa⁺⁺ measurements (fura-2) in response to 5 nM GSK101. Note the increasein signal in response to co-exposure to ET1. (B) ET1 secretion bynon-stimulated 1° MK depends on TRPV4 and PLC. Shown are relative ET1concentrations (determined by ELISA, pg/mL; vehicle-treated and WTcontrol normalized to 100) in supernatant of non-stimulated 1° MK. Notethe clear dependence on TRPV4, as indicated by a 50% reduction inTrpv4^(−/−) 1° MK. Moreover, there is a significant down-regulation byspecific inhibition of TRPV4, which is dose-dependent (two doses ofGSK205) and can be mediated by two different compounds (GSK205, RN1734).There is also down-regulation of ET1 secretion by a specific inhibitorof PLC (U73122), and by an ET-convertase inhibitor, CGS35066, whichserved as a control compound. In addition, PLC-inhibitor robustlyaffects ET1 secretion in WT and Trpv4^(−/−) 1° MK. (C) ET1 expression byUVB-exposed 1° MK depends on TRPV4 and PLC—immunocytochemistry. Shown isspecific ET1 immunolabeling in 1° MK, exposed to UVB using the UVB-LEDs,as for Ca⁺⁺ imaging. Use of the UVB-LED device precluded application ofa ET1 ELISA, only irradiated cells could be examined. Note thesignificant down-regulation of ET1 immunoreactivity by specificinhibition of TRPV4 (two different compounds, GSK205, RN1734), by PLCinhibition (U73122), also by inhibition of ET-convertase (CGS35066). (D)ET1 expression by UVB-exposed 1° MK depends on TRPV4 andPLC—quantification of immunocytochemistry. Densitometric measurements ofn≥25 cells per condition, background subtracted, are shown, indicating asignificant upregulation of ET1 in response to UVB (* p<0.05 ANOVA), andsignificant down-regulation vs. control-treated and UVB-exposed cellsfor treatments with selective TRPV4 antagonists (GSK205, RN1734),PLC-inhibitor U73122 and ET-convertase inhibitor CGS35066; * p<0.05,t-test; # p<0.05 ANOVA.

FIG. 9: UVB-evoked inflammasome activation in keratinocytes depends onTRPV4. (A) Caspase-1 immunolabeling in footpad skin in response to UVB.Representative images are from sections of skins before stimulation(control) or 48 hours post-UVB exposure. Bars=20 μm. (B) Quantificationsof caspase-1 immunolabeling. Bar diagrams show densitometry, n≥3animals/group. Comparisons: UVB exposed WT vs. Trpv4−/− and iKO+oil vs.iKO+tam. **p<0.01 ANOVA. (C) Western blotting for caspase-1 from 1°MK±UVB-exposure. Note that caspase-1 levels, in particular cleavedcaspase-1 (lower band), are elevated in UVB-exposed WT cells, but thereis a complete absence of both procaspase-1 and cleaved caspase-1 in 1°MK from Trpv4−/− mice. (D) IL-1β is induced upon UVB-exposure and isdependent on TRPV4. Anti-IL-1β immunofluorescence, otherwise as in panelA. (E) Quantifications of IL-1β immunolabeling, n≥3 animals/group.Comparisons: UVB exposed WT vs. Trpv4−/− and iKO+oil vs. iKO+tam, **p<0.01 ANOVA. (F) IL-1β concentrations in interstitial fluid ofUVB-exposed footpad. IL-1β levels (ELISA) are shown in lavagedinterstitial fluid. Note strong up-regulation in WT and oil-treated iKOmice after UVB, in contrast significant attenuation in Trpv4−/− andtam-treated iKO mice. n≥5 mice/group, ** p<0.01 ANOVA. (G) CXCL5 isinduced upon UVB-exposure and is dependent upon TRPV4. Anti-CXCL5immunolabeling, otherwise as in panel A. (H) Quantifications of CXCL5immunolabeling, n≥3 animals/group. Comparisons: UVB exposed WT vs.Trpv4−/− and iKO+oil vs. iKO+tam, ** p<0.01 ANOVA.

FIG. 10: Epidermal TRPV4, ET1 and IL-1β are elevated in photodermatitisas compared to healthy human skin. (A) Representative micrographs ofTRPV4, ET1 and IL-1β distribution in the epidermis of acutephotodermatitis, as compared to healthy human skin. Immunostaining foreach antigen is increased in acute photodermatitis vs. healthy skin.Scale-bars=50 μm (left), 100 μm (middle), 50 μm (right). (B)Morphometric analysis for immunoreactive TRPV4, ET1 and IL-1β. Findingsreveal significantly increased immunolabeling for all three proteins inacute photodermatitis as compared to healthy human skin (n=3 subjectsfor normal, healthy skin, and 3 patients for acute UV photodermatitis).

FIG. 11: Exemplars of human chronic photodermatitis. Upper panel showsnormal healthy human skin, as displayed in FIG. 10A, for comparison.Lower panel shows examples of chronic photodermatitis with elevatedexpression of TRPV4, in spinous and basal layers, ET1 (throughout) andIL-1β (throughout). In comparison to acute photodermatitis, note reducedinterstitial intraepidermal edema. Bar=50 μm.

FIG. 12: External-topical application of a selective TRPV4 inhibitorattenuates UVB-evoked nocifensive behavior and inflammation. (A)UVB-induced nocifensive behavior. Pain behavior is attenuated by topicalapplication of GSK205. The left-hand diagram shows withdrawal thresholdsafter UVB-exposure in response to noxious thermal cues (Hargreaves'test), and their modulation by two doses of topically applied GSK205 (1mM and 5 mM; applied 60′ and 10′ pre-exposure). The higher dose led to asignificant attenuation of thermal allodynia at 48 hours post-UVB; n=6mice/group; ** p<0.01 ANOVA. The righthand diagram shows development ofmoderate thermal allodynia in Trpv4−/− mice, and similar sensitizationfor vehicle-treated vs. 5 mM GSK205-treated mice, indicating lack ofoff-target effects of the compound at 5 mM; n=5 mice/group. (B)GSK205-treatment attenuates keratinocyte expression of IL-1β inUVB-exposed footpad—representative micrographs. Bars=20 μm. (C)GSK205-treatment attenuates keratinocyte expression of IL-1β inUVB-exposed footpad quantifications. Bar diagrams show densitometryresults from n=3 mice/group, ** p<0.01 ANOVA. (D) GSK205-treatmentattenuates secretion of IL-1β by UVB-exposed 1° MK. IL-1β concentrationsin supernatant (ELISA), are shown in response to UVB. Cells werecultured +/−5 μM GSK205. Note prevention of increase in IL-1β secretionin response to UVB upon treatment with GSK205. ** P<0.01 ANOVA.

FIG. 13: Topical application of a selective TRPV4 inhibitor attenuatesUVB-evoked nocifensive behavior by suppressing upregulation ofpro-algesic/algogenic mediators in murine keratinocytes—Findings forCXCL5 and IL6. (A) GSK205-treatment attenuates keratinocyte expressionof CXCL5 in UVB-exposed footpad—micrographs and quantitation. As in FIG.12B, specific immunolabeling for CXCL5, which is selectively upregulatedin footpad keratinocytes in response to UVB, note attenuation withGSK205 treatment. Bar diagrams show densitometry of CXCL5immunolabeling, n=3 animals per group, ≥3 sections analyzed per animal.Note significant differences for vehicle-treated mice betweenUVB-exposed and non-exposed, no such difference for mice treatedtopically with 5 mM GSK205. Comparison UVBexposed between vehicle andGSK205-treated, ** p<0.01 ANOVA. Bar=20 μm. (B) GSK205-treatmentattenuates keratinocyte expression of IL-6 in UVB-exposedfootpad—micrographs and quantification. As in FIG. 12B and FIG. 13A,specific immunolabeling for IL-6, demonstrates similar regulation ofIL-6 as for CXCL5 and IL-1β. Comparison UVB-exposed between vehicle andGSK205-treated, ** p<0.01 ANOVA. Bar=20 μm.

FIG. 14: External-topical application of a selective TRPV4 inhibitorattenuates UVB-evoked nocifensive behavior and inflammation. (A)UVB-photodermatitis is attenuated in mice treated with GSK205.Representative H&E micrographs of paw-pad skin are shown, bars=20 μm.Treatment of UVB-exposed skin with GSK205 improved the skin architecturein mice as compared to vehicle-treated mice after 24 hours. (i) and(iii) Representative skin sections of UVB-induced photodermatitis afterGSK205 treatment showed markedly reduced inflammatory infiltrate, lessspongiosis and dermal-epidermal blisters with remaining epidermalthickening. (ii) and (iv) Vehicle-treated mice after UVB-inducedphotodermatitis were characterized by signs of severe acutephotodermatitis such as spongiosis, epidermal hyperkeratosis, disrupteddermal-epidermal border (blister), and a marked inflammatory infiltratewith dilated blood vessels and dermal edema (arrows). Also note theerythrocyte accumulation in blood vessels indicative of dermatitis. (B)Topical treatment with a TRPV4-specific inhibitor attenuatesupregulation of algogenic ET1/Edn1. Edn1 mRNA was determined by qPCRafter extraction of total RNA from paw-pad epidermis. In vehicle treatedskin, note increase of Edn1 expression with early up-regulation at the 2hour time-point, and sustained elevation up to the 24 hour time-point.This time-course resembles that seen in WT control mice, when comparingto Trpv4−/−(FIG. 15). Importantly, topical treatment with 5 mM GSK205results in complete lack of this regulation; n=4 mice/group, *p<0.05ANOVA. (C) GSK205 does not function as sunscreen. Schematic illustratesthe experimental set-up. (D) GSK205 does not function as sunscreen. Thebar diagram shows results from n=7-8 mice/group, note absence of achange in UVB permeation with 5 mM GSK205, topically applied as for (B),vs. vehicle control, yet significantly reduced with sunscreen SPF100.

FIG. 15: Upregulation of ET1/End1 in mouse paw in response to UVB. Edn1mRNA was determined by qPCR after isolation of total RNA from paw-padepidermis. Note the early increase, at the 2 hour time-point, in WTcontrol epidermis, and its complete lack in Trpv4−/−, and sustainedupregulation at 24 hours, slightly reduced vs. 2 hour time-point, againcomplete lack of upregulation in Trpv4−/−. Quantifications are for n=4mice/group. ** denotes statistically significant (p<0.01, t-test).

FIG. 16: Skin UVB permeability testing. (A) Experimental set-up fortesting of skin permeability to UVB. (B) Results from A. Note thatintensity is 70% within 500 μm radius to the center of the UV beam.

FIG. 17: Role of TRPV4 in itch transmission in mice in vivo. Histamine(10%) was injected intracutaneously into the cheek of C57b/6 control(WT) or TRPV4 knockout mice (Trpv4 pan-knockout; n=6 per group). Over 30min, TRPV4 null mice showed a significant reduction ((p<0.01) t-test))of itch-behavior as compared to WT mice.

FIG. 18: The role of TRPV4 in itch. Shown is a graph of scratchingbehavior after administration of a pruritogen in mice with TRPV4deletion in keratinocytes after induction of the TRPV4 knockout, ascompared to mice without induction. Without induction, the mice functionas wild-type control mice (Moore et al. Proc. Natl. Acad. Sci. U.S.A.2013, 110, E3225-E3234). Compared to control mice, scratch behavior wassignificantly reduced for mice in which TRPV4 channels had beenselectively deleted in skin keratinocytes.

FIG. 19: Compound 16-8/18hy. Compound 16-8/18h was designed as a hybridof compounds 16-8 and 16-18.

FIG. 20: Inhibition of calcium ion flux through TRPV4. Compounds of thepresent invention demonstrated inhibitory activity against TRPV4 andwere stronger antagonists that GSK205.

FIG. 21: Treatment of pain. Compounds as disclosed herein attenuatednocifensive behavior in mice.

FIG. 22: Treatment of pain after UVB exposure. Compounds as disclosedherein attenuated nocifensive behavior in a mouse model for sunburn.

FIG. 23: Effect on TRPA1. Compounds as disclosed herein inhibited TRPA1,as indicated by measuring calcium transience.

FIG. 24: Effect on TRPV1, TRPV2, and TRPV3. Compounds as disclosedherein did not inhibit TRPV1, TRPV2, or TRPV3, as indicated by measuringcalcium transience.

FIG. 25: Shown are graphs of calcium transience in mammalian epidermalkeratinocytes in the presence of activators for histamine receptors,with (bottom curves) or without (top curves) the TRPV4 inhibitor GSK205.

DETAILED DESCRIPTION

In a broad sense, the disclosure relates to compositions and methods fortreating and/or preventing a dermatological disorder. The skin functionsas an essential barrier between the external environment and thevertebrate organism. Keratinocytes in the skin absorb UV-light, leadingto skin inflammation, pain, and itch after over-exposure, subsequentlyto skin pigmentation. The inventors have identified that the skin, inparticular its epidermal epithelia, is more substantially involved insensory transduction. For this, the inventors used a mouse model ofsunburn in order to induce a state of lowered sensory thresholds evokedby a limited, self-resolving inflammation in response to UV spectrum oflight. UV-evoked lowering of sensory thresholds shares major hallmarksof pathological pain, which is another valuable feature of this model.

The compositions and methods disclosed herein relate to theidentification and characterization that epidermal keratinocytesfunction prominently to orchestrate UVB-mediated inflammation andsensitization of peripheral nerve endings in the skin, and in thatrespect, epidermal keratinocytes have a role similar to a co-sensorycell. Keratinocytes abundantly express the cation channel protein TRPV4,and the inventors have determined that TRPV4, expressed in epidermalkeratinocytes, plays a role in UV-induced inflammation and pain andother inflammatory and/or dermatological disorders. The channel exertsits role as a master regulator of UVB-evoked skin inflammation andnociception through Ca++ influx into keratinocytes. This UVB-evoked,TRPV4-mediated Ca++ influx re-programs the keratinocyte to function in apro-inflammatory and pro-algesic (pro-pain) manner, via TRPV4-dependentsecretion of endothelin-1, which may lead to sensation of itch and skinpigmentation. TRPV4 is activated contemporaneously with UVB exposure,which leads to activation of pro-algesic pathways via secreted factorspreviously demonstrated to have relevance in human pain. As disclosed infurther detail herein, mice with inducible Trpv4 deletions targeted tokeratinocytes were induced for TRPV4 deletion, subsequently UVB-exposed,and found to be less sensitive to noxious thermal and mechanicalstimulation than control mice. Epidermal TRPV4 was identified as aprotein involved in the orchestration of UVB-mediated skin inflammation.In mouse skin, UVB-evoked inflammasome activation and increasedexpression of pro-algesic/algogenic mediators, such as IL1-β, CXCL5,ET-1, and IL-6, were TRPV4-dependent. ET-1 has been shown in humans tonot only elicit painful sensations, but to also elicit itch, wheninjected into the skin. Also, ET-1 has been identified as a melanogen,that is, to increase skin pigmentation by signaling to melanocytes. Inprimary murine keratinocytes, UVB caused a direct, TRPV4-dependentCa⁺⁺-response. Moreover, in mice, topical application of aTRPV4-selective inhibitor reduced UVB-evoked epidermal inflammation andpain behavior. Additionally, it was found that epidermal expression ofTRPV4, ET1, and IL1β were increased in acute human UV-photodermatitis.The term photodermatitis is used in this application referring to skininflammation in response to UV radiation/light. This tissue response caninclude pain, irritation, itch, influx of inflammatory andpain-enhancing cells and tissue injury. The compounds as detailed hereinmay inhibit TRPA1. The compounds as detailed herein may inhibit TRPV4and TRPA1. The compounds as disclosed herein may not inhibit TRPV1,TRPV2, or TRPV3. The inhibitor may specific for TRPV4 and TRPA1.

The dermatological disorder may be associated with the TRPA1 or TRPV4pathway. Dermatological disorders include, but are not limited to,photo-induced inflammation, pain in diseases involving skin pain, itch,cancer, autoimmune diseases, fibrotic diseases, other acneiform orinflammatory skin diseases, and pigmentation disorders. For example,dermatological disorders may include, but are not limited to, sunburn;photoallergic reaction; phototoxic reaction; phytophotodermatitis(Berloque dermatitis); acute and chronic actinic dermatitis; atopicdermatitis exacerbation; all subtypes of rosacea includingtrigeminal-pain associated rosacea; all lupus erythematosus subtypes(systemic, discoid, subacute); atopic dermatitis; actinic prurigo;prurigo nodularis; prurigo subacuta; prurigo pigmentosa; Lichen simplex(also called neurodermatitis); diabetic pruritus; uremic pruritus;pruritus induced by metabolic (liver) diseases; pruritus induced bymalignancies like lymphoma; pruritus induced by polycythemia vera;pruritus induced by scabies; pruritus induced by bullous pemphigoid;pruritus induced by urticaria (especially but not exclusively actinicurticaria); pruritus induced by insect/arachnoid vector bite; pruritusinduced by parasitosis; melanoma; non-melanoma skin cancer (BCC, SCC);actinic keratosis and other premalignant skin cancers; mycosisfungoides; Sézary syndrome; Xeroderma pigmentosum; Cockayne syndrome;all lupus erythematosus subtypes (systemic, discoid, subacute);dermatomyositis; erythema multiforme; lichen planus; fibrotic diseasesinduced by UV-exposure (Rhinophyma, chronic actinic dermatitis, actinicreticuloid, photoaging, hyalinosis cutis et mucosae; polymorph lighteruption; Acne aestivalis; all porphyria subforms with implications onphoto-induced skin changes (erythropoetic porphyria, erythropoeticprotoporphyria, Porphyria variegate); photo-induced Herpes simplexinfection (Herpes labialis); morbus Darier; disseminated superficialactinic porokeratosis; pityriasis rubra pilaris; Bloom syndrome;Rothmund-Thomson syndrome; Hartnup syndrome photoaging; wrinkles;photo-induced inflammation; pathologic skin fibrotic response; synovialfibrosis; lung fibrosis; keloid formation; chemical or actinic injury; apathologic response to skin irradiation (“radio-derm”); psoriasis;chronic atopic dermatitis; chronic UV-dermatitis; scleroderma;conditions characterized by a chronic maladaptive injury response to anongoing noxious insult to the skin; inappropriate scar formation,pigmentation; and pigmentation disorders.

Methods

In an aspect, the disclosure provides methods of reducing skininflammation in a subject in need thereof. The methods may compriseadministering to the subject an effective amount of a TRPA1 and/or TRPV4inhibitor. The skin inflammation may be related to UVB exposure.

Skin inflammation may be associated with conditions including, but notlimited to, sunburn (acute photodermatitis), photoallergic reaction,phototoxic reaction, phytophotodermatitis (Berloque dermatitis), acuteand chronic actinic dermatitis, atopic dermatitis exacerbation, androsacea.

In other aspects, the disclosure provides methods of pain management.The methods may comprise administering to at least a portion of the skinof a subject in need thereof an effective amount of a TRPA1 and/or TRPV4inhibitor. The pain may be related to UVB exposure.

Pain may be chronic or acute. Pain may be associated with or result fromconditions including, but not limited to, all subtypes of rosaceaincluding trigeminal-pain associated rosacea, reflex sympatheticdystrophy (RSD), and all lupus erythematosus subtypes (systemic,discoid, subacute).

In other aspects, the disclosure provides methods of reducing itch in asubject in need thereof. ET-1 has been shown to elicit itch, and asshown in the Examples, increased expression of ET-1 was TRPV4-dependent.The methods may comprise administering to the subject an effectiveamount of a TRPA1 and/or TRPV4 inhibitor.

Itch may be chronic or acute. Itch may be associated with or result fromconditions including, but not limited to, rosacea, atopic dermatitis,actinic prurigo, prurigo nodularis, prurigo subacuta, prurigopigmentosa, Lichen simplex (also called neurodermatitis), diabeticpruritus, and uremic pruritus. Itch or pruritus may be associated withor result from conditions including metabolic (liver) diseases,malignancies like lymphoma, polycythemia vera, scabies, bullouspemphigoid, urticaria (especially but not exclusively actinicurticaria), insect/arachnoid vector bite, and parasitosis.

In other aspects, the disclosure provides methods of treating cancer ina subject in need thereof. The methods may comprise administering to thesubject an effective amount of a TRPA1 and/or TRPV4 inhibitor.

The cancer and related conditions may include, but are not limited to,melanoma, non-melanoma skin cancer (BCC, SCC), actinic keratosis andother premalignant skin cancers, mycosis fungoides, Sézary syndrome, andXeroderma pigmentosum.

In other aspects, the disclosure provides methods of treating anautoimmune disease in a subject in need thereof. The methods maycomprise administering to the subject an effective amount of a TRPA1and/or TRPV4 inhibitor.

Autoimmune diseases may include, but are not limited to, all lupuserythematosus subtypes (systemic, discoid, subacute), dermatomyositis,erythema multiforme, and lichen planus.

In other aspects, the disclosure provides methods of treating a fibroticdisease in a subject in need thereof. The methods may compriseadministering to the subject an effective amount of a TRPA1 and/or TRPV4inhibitor.

Fibrotic diseases may include conditions induced by UV-exposure, suchas, for example, Rhinophyma, chronic actinic dermatitis, actinicreticuloid, photoaging, and hyalinosis cutis et mucosae. Fibroticdisease may include at least one disorder or condition selected frompathologic skin fibrotic response, synovial fibrosis, and lung fibrosis.Pathologic skin fibrotic response may include at least one disorder orcondition selected from skin fibrosis, keloid formation, chemical oractinic injury, a pathologic response to skin irradiation(“radio-derm”), psoriasis, chronic atopic dermatitis, chronicUV-dermatitis, scleroderma, and conditions characterized by a chronicmaladaptive injury response to an ongoing noxious insult to the skin orinappropriate scar formation.

As detailed in the Examples, secretion of periostin (93 kDa protein) bykeratinocytes in response to histamine exposure is dependent on TRPV4.Periostin was detected in the histamine-stimulated cells, but theprotein was not detectable in vehicle-stimulated cultures, and GSK205completely eliminated secretion of periostin. Periostin has a role inchronic allergic skin inflammation and also pathologic-fibroticremodeling of the skin. Histamine activates TRPV4 channels via specificactivation of histamine H1R-receptors in keratinocytes. ThisTRPV4-dependent calcium influx regulates release of periostin fromkeratinocytes. In terms of fibrotic tissue remodeling of organs, TRPV4channels expressed by fibroblasts may have a role in lung fibrosisevoked by bleomycin, which may relate to fibroblasts elsewhere,including skin fibroblasts and synovial fibroblasts. Inhibitors asdetailed herein may potently block TRPV4 activation in keratinocytes andalso skin fibroblasts. Based on the above signaling mechanisms, thiswill attenuate and prevent fibrotic responses in the skin, also chronicskin-remodeling tissue responses mediated by endogenous pathogeneticmediator molecules such as periostin. As for synovia of the joint, oneresponse to joint trauma is development of synovial fibrosis andcontracture. Synoviocytes, synovial fibroblasts and infiltratinginflammatory cells may have roles in this maladaptive response, as allof these cells are known to express TRPV4.

In other aspects, the disclosure provides methods of treating otheracneiform or inflammatory skin disease in a subject in need thereof. Themethods may comprise administering to the subject an effective amount ofa TRPA1 and/or TRPV4 inhibitor.

Acneiform or inflammatory skin diseases may include, but are not limitedto, polymorph light eruption, Acne aestivalis, photo-induced Herpessimplex infection (Herpes labialis), morbus Darier, disseminatedsuperficial actinic porokeratosis, Pityriasis rubra pilaris, and allporphyria subforms with implications on photo-induced skin changes suchas, for example, erythropoetic porphyria, erythropoetic protoporphyria,and Porphyria variegate.

In other aspects, the disclosure provides methods of reducing skinpigmentation in a subject in need thereof. ET-1 has been shown to signalto skin melanocytes and function as a major melanogen (=enhancing skinpigmentation), and as shown in the Examples, increased expression ofET-1 was TRPV4-dependent. The methods may comprise administering to thesubject an effective amount of a TRPA1 and/or TRPV4 inhibitor.

Skin inflammation, pain, itch, and/or pigmentation may also beassociated with disorders including, but not limited to, Cockaynesyndrome, non-UV skin burn less than 3^(rd) degree, disturbed woundhealing, exposure and pathological response to poison ivy, and pain ofbone fractures directly adjacent to the skin such as fractures of thetibia, digits, or skull. For example, one or more of these disorders maybe symptomatic of reflex sympathetic dystrophy (RSD).

In other aspects, the disclosure provides methods of preventingdermatological diseases or disorders such as irritation, pain, itch,pruritus, autoimmune diseases, skin cancer (including melanoma, forexample, with topical treatment of TRPA1 and/or TRPV4 inhibitor-based UVprotection), autoimmune diseases, fibrotic disorders, pigmentationdisorders, and others as described above. In some embodiments, thedisclosure provides methods of preventing the development and/orexacerbation of Xeroderma pigmentosum, Cockayne syndrome, Bloomsyndrome, Rothmund-Thomson syndrome, and Hartnup syndrome.

In other aspects, the disclosure provides methods of treating orpreventing cosmetic conditions. For example, the disclosure providesmethods of treating or preventing photoaging, wrinkles, photo-inducedinflammation, pigmentation, and pigmentation disorders.

TRPA1 and/or TRPV4 Inhibitor

As TRPV4 is a Ca²⁺-permeable, nonselective cation channel, someembodiments provide for a TRPA1 and/or TRPV4 inhibitor that can inhibitthe biological function of TRPA1 and/or TRPV4 (e.g., inhibit cationchannel activity, inhibit Ca++ transport and/or availability). Otherembodiments provide for a TRPA1 and/or TRPV4 inhibitor that may inhibitthe expression of mRNA encoding TRPA1 or TRPV4. Some embodiments providea TRPA1 and/or TRPV4 inhibitor that may inhibit the translation of mRNAencoding TRPA1 or TRPV4 to protein. Thus, a TRPA1 and/or TRPV4 inhibitormay indirectly or directly bind and inhibit the activity of TRPA1 and/orTRPV4 (e.g., binding activity or enzymatic activity), reduce theexpression of TRPA1 and/or TRPV4, prevent expression of TRPA1 and/orTRPV4, or inhibit the production of TRPA1 and/or TRPV4 in a cell.Inhibit or inhibiting relates to any measurable reduction or attenuationof amounts or activity, e.g., amounts or activity of TRPA1 and/or TRPV4,such as those disclosed herein.

In some embodiments, a TRPA1 and/or TRPV4 inhibitor can increase theamount of, or the biological activity of a protein that can reduce theactivity of TRPA1 and/or TRPV4. Agents capable of increasing the levelof such a protein may include any agent capable of increasing protein ormRNA levels or increasing the expression of the protein. In oneembodiment, a TRPA1 and/or TRPV4 inhibitor may comprise the proteinitself. For example, a TRPA1 and/or TRPV4 inhibitor may includeexogenously expressed and isolated protein capable of being delivered tothe cells. The protein may be delivered to cells by a variety ofmethods, including fusion to Tat or VP16 or via a delivery vehicle, suchas a liposome, all of which allow delivery of protein-based agentsacross the cellular membrane. Those of skill in the art will appreciatethat other delivery mechanisms for proteins may be used. Alternatively,mRNA expression may be enhanced relative to control cells by contactwith a TRPA1 and/or TRPV4 inhibitor. For example, the agent capable ofincreasing the level of natively expressed protein may include a geneexpression activator or de-repressor. As another example, a TRPA1 and/orTRPV4 inhibitor capable of decreasing the level of natively expressedprotein may include a gene expression repressor. An agent capable ofincreasing the level of protein may also include agents that bind todirectly or indirectly and increase the effective level of protein, forexample, by enhancing the binding or other activity of the protein. Theagent capable of decreasing the level of protein may also include agentsthat bind to directly or indirectly and decrease the effective level ofprotein, for example, by inhibiting or reducing the binding or otheractivity of the protein.

The amount or level of expression of a biomolecule (e.g., mRNA orprotein) in a cell may be evaluated by any variety of techniques thatare known in the art. Thus, inhibit or inhibiting, such as, for example,the level of protein expression (e.g., TRPA1 and/or TRPV4), may beevaluated at either the protein or mRNA level using techniquesincluding, but not limited to, Western blot, ELISA, Northern blot, realtime PCR, immunofluorescence, or FACS analysis. For example, theexpression level of a protein may be evaluated by immunofluorescence byvisualizing cells stained with a fluorescently-labeled protein-specificantibody, Western blot analysis of protein expression, and RT-PCR ofprotein transcripts. The expression level of TRPA1 and/or TRPV4 may becompared to a control. A control may include comparison to the level ofexpression in a control cell, such as a non-disease cell or other normalcell. Alternatively a control may include an average range of the levelof expression from a population of normal cells. Alternatively, astandard value developed by analyzing the results of a population ofcells with known responses to therapies or agents may be used. Thoseskilled in the art will appreciate that a variety of controls may beused.

A TRPA1 and/or TRPV4 inhibitor may comprise a variety of compounds andcompositions and agents. For example, a TRPA1 and/or TRPV4 inhibitor maycomprise a compound. A TRPA1 and/or TRPV4 inhibitor may comprise abiological molecule, including nucleic acid molecules, such as apolynucleotide having RNAi activity against TRPA1 and/or TRPV4 or asubstrate thereof. In embodiments, the nucleic acid molecules caninclude decoy RNAs, dsRNAs, miRNAs, siRNAs, nucleic acid aptamers,antisense nucleic acid molecules, and enzymatic nucleic acid moleculesthat comprise a sequence that is sufficient allow for binding to anencoding nucleic acid sequence and inhibit activity thereof (i.e., arecomplementary to such encoding nucleic acid sequences). Suitably, anRNAi molecule comprises a sequence that is complementary to at least aportion of a target sequence such that the RNAi can hybridize to thetarget sequence under physiological or artificially defined (e.g.,reaction) conditions. In some embodiments an RNAi molecule comprises asequence that is complementary such that the molecule can hybridize to atarget sequence under moderate or high stringency conditions, which arewell known and can be determined by one of skill in the art. In someembodiments an RNAi molecule has complete (100%) complementarity overits entire length to a target sequence. A variety of RNAi molecules areknown in the art, and can include chemical modifications, such asmodifications to the sugar-phosphate backbone or nucleobase that areknown in the art. The modifications may be selected by one of skill inthe art to alter activity, binding, immune response, or otherproperties. In some embodiments, the RNAi can comprise an siRNA having alength from about 18 to about 24 nucleotides.

In some embodiments, the inhibitory nucleic acid molecule can bind to atarget nucleic acid sequence under stringent binding conditions. Theterms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a polynucleotide willhybridize to its target sequence, to a detectably greater degree thanother sequences (e.g., at least 2-fold over background). An example ofstringent conditions include those in which hybridization in 50%formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to65° C. is performed. Amino acid and polynucleotide identity, homologyand/or similarity can be determined using the ClustalW algorithm,MEGALIGN™ (Lasergene, WI). Given a target polynucleotide sequence, forexample of TRPA1 and/or TRPV4 or biological substrate thereof, aninhibitory nucleic acid molecule can be designed using motifs andtargeted to a region that is anticipated to be effective for inhibitoryactivity, such as is known in the art.

In other embodiments, a TRPA1 and/or TRPV4 inhibitor comprises anantibody that can specifically bind to a protein such as TRPA1 and/orTRPV4 or a fragment thereof. Embodiments also provide for an antibodythat inhibits TRPA1 and/or TRPV4 through specific binding to a TRPA1and/or TRPV4 substrate molecule. The antibodies can be produced by anymethod known in the art, such as by immunization with a full-lengthprotein such as TRPA1 and/or TRPV4, or fragments thereof. The antibodiescan be polyclonal or monoclonal, and/or may be recombinant antibodies.In embodiments, antibodies that are human antibodies can be prepared,for example, by immunization of transgenic animals capable of producinga human antibody (see, for example, International Patent ApplicationPublication No. WO 93/12227). Monoclonal antibodies (mAbs) can beproduced by a variety of techniques, including conventional monoclonalantibody methodology, e.g., the standard somatic cell hybridizationtechnique of Kohler and Milstein, and other techniques, e.g., viral oroncogenic transformation of B-lymphocytes. Animal systems for preparinghybridomas include mouse. Hybridoma production in the mouse is very wellestablished, and immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are well known in the art. Fusionpartners (e.g., murine myeloma cells) and fusion procedures are alsoknown.

Any suitable methods can be used to evaluate a candidate active agentfor inhibitory activity toward TRPA1 and/or TRPV4. Such methods caninclude, for example, in vitro assays, in vitro cell-based assays, exvivo assays, and in vivo methods. The methods can evaluate bindingactivity, or an activity downstream of the enzyme of interest. Ex vivoassays may involve treatment of cells with an agent of the invention,followed by detection of changes in transcription levels of certaingenes, such as TRPA1 and/or TRPV4 through collection of cellular RNA,conversion to cDNA, and quantification by quantitative real timepolymerase chain reaction (RT-QPCR). Additionally, the cell viability orinflammation may be determined after treatment with an agent.

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is according toFormula I:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups;

D is C₁-C₃ alkylene;

E is a bond, or C₁-C₂ alkylene; and

R is selected from the group consisting of hydrogen, hydroxyl, amino,alkyl, alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring. Insome embodiments, B and C are independently a phenyl group. In someembodiments, A is phenyl or heteroaryl. In some embodiments, A ispyridnyl. In some embodiments, R is C1-C4 alkyl. In some embodiments, Ais heteroaryl, B and C are phenyl, D is ethylene, E is methylene, and Ris methyl. In some embodiments, R is ethyl.

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor excludes thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In some embodiments, the disclosure provides a method of treating asubject wherein the method comprises administering an inhibitor of TRPA1and/or TRPV4 in a pharmaceutically acceptable composition.

In other aspects, the disclosure provides compositions comprising aTRPA1 and/or TRPV4 inhibitor.

“Administration” or “administering” refers to delivery of a compound orcomposition by any appropriate route to achieve the desired effect.Administration may include any convenient route of administration,whether systemically/peripherally or at the site of desired action,including but not limited to, oral (e.g. by ingestion); topical(including e.g. transdermal, intranasal, ocular, buccal, andsublingual); pulmonary; respiratory (e.g. by inhalation or insufflationtherapy using, e.g. an aerosol, e.g. through mouth or nose); rectal;vaginal; parenteral, for example, by injection, including subcutaneous,intradermal, intramuscular, intravenous, intraarterial, intracardiac,intrathecal, intraspinal, intracapsular, subcapsular, intraorbital,intraperitoneal, intratracheal, subcuticular, intraarticular,subarachnoid, and intrasternal; by implant of a depot, for example,subcutaneously or intramuscularly. In certain embodiments,administration may be topical. “Co-administered” refers to simultaneousor sequential administration. A compound or composition may beadministered before, concurrently with, or after administration ofanother compound or composition. TRPV4 and/or TRPA1 inhibitors can beapplied topically to joints, either with penetration-enhancing deliveryvehicles (knee and ankle, finger joints, elbow, temporomandibularjoint), or by targeted injection (hip, shoulder, spinal). Topicalapplication may be used for the cornea of the eye to prevent scarringand fibrotic response in this sensitive spot. For prevention of lungfibrosis, e.g. upon treatment with bleomycin, topical application byinhalation of the TRPV4 and/or TRPA1 inhibitors may be used foreffective treatment of lung fibrosis.

One skilled in the art can select an appropriate dosage and route ofadministration depending on the patient, the particular disease,disorder, or condition being treated, the duration of the treatment,concurrent therapies, etc. In certain embodiments, a dosage is selectedthat balances the effectiveness with the potential side effects,considering the severity of the disease, disorder, or condition (e.g.,skin inflammation, pain, or itch).

“Pharmaceutically acceptable” means suitable for use in a human or othermammal. The terms “pharmaceutically acceptable carriers” and“pharmaceutically acceptable excipients” are used interchangeably andrefer to substances that are useful for the preparation of apharmaceutically acceptable composition. In certain embodiments,pharmaceutically acceptable carriers are generally compatible with theother ingredients of the composition, not deleterious to the recipient,and/or neither biologically nor otherwise undesirable.

The composition may comprise the TRPA1 and/or TRPV4 inhibitor incombination with a carrier, vehicle, or diluent. Embodiments provide forpharmaceutically acceptable carriers including, but not limited to,substances useful for topical, intrathecal, ocular, parenteral,intravenous, intraperitoneal intramuscular, sublingual, nasal, and oraladministration. Administration may be systemic. “Pharmaceuticallyacceptable carrier” also includes agents for preparation of aqueousdispersions and sterile powders for injection or dispersions. Examplesof pharmaceutically acceptable carriers and excipients are discussed,e.g., in Remington Pharmaceutical Science, 16th Ed. Certain exemplarytechniques and compositions for making dosage forms are described in thefollowing references: Modern Pharmaceutics, Chapters 9 and 10, Banker &Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms:Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms,2nd Ed., (1976). The carrier, vehicle, or diluent may be suitable fortopical application.

In certain embodiments, compositions are formulated for topicaladministration. For compositions suitable for topical administration,the composition may be combined with one or more carriers and used inthe form of cosmetic formulations. Formulations may include a foam,cream, gel, lotion, ointment, or solution. For example, a TRPA1 and/orTRPV4 inhibitor may be suitably dissolved in the alcohol of skindisinfectant gel or in lotions, creams, or other formulations. Incertain embodiments, a TRPA1 and/or TRPV4 inhibitor may be included inor added to a cosmetic formulation. In certain embodiments, a TRPA1and/or TRPV4 inhibitor may be included in or added to sun protectiontopical formulations.

For oral therapeutic administration, the composition may be combinedwith one or more carriers and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,chewing gums, foods, and the like. The percentage of the compositionsand preparations may, of course, be varied and may conveniently bebetween about 0.1 to about 100% of the weight of a given unit dosageform. The tablets, troches, pills, capsules, and the like may alsocontain the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, fructose, lactose or aspartame or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring. Theabove listing is merely representative and one skilled in the art couldenvision other binders, excipients, sweetening agents and the like. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like.

“Effective amount” refers to a dosage of a compound or compositioneffective for eliciting a desired effect, commensurate with a reasonablebenefit/risk ratio. This term as used herein may also refer to an amounteffective at bringing about a desired in vivo effect in an animal,preferably, a human, such as reduction in skin inflammation, reductionin pain, or reduction in itch.

The amount of a TRPA1 and/or TRPV4 inhibitor in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained. The selected dosage level will depend upon a variety offactors including the activity of the particular compound employed, theroute of administration, the time of administration, the rate ofexcretion or metabolism of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compound employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

In general, the daily dose contains from about 0.1 mg to about 2000 mgof the active ingredient, or about 0.5 to about 60 mg of the activeingredient. This dosage form permits the full daily dosage to beadministered in one or two oral doses. More than once daily or twicedaily administrations, e.g., 3, 4, 5, or 6 administrations per day, arealso contemplated herein.

In some embodiments, as noted above, administering relates to providingan amount effective at bringing about a desired in vivo effect such asinhibition of TRPA1 and/or TRPV4 in an animal, such as a human. As usedherein, a “subject in need of treatment” refers to a subject having beendiagnosed with a dermatological disease or disorder associated with skininflammation, pain, itch, or a combination thereof. A subject may be amammalian subject. In embodiments a subject can include human andnon-human animals. The term “non-human animals” includes allvertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles)and mammals, such as non-human primates, domesticated and/oragriculturally useful animals (such as sheep, dogs, cats, cows, pigs,etc.), and rodents (such as mice, rats, hamsters, guinea pigs, etc.).Accordingly, embodiments of the methods described herein relate totreatment of a cell or tissue, a cell or tissue from a subject, or asubject that may be a eukaryote, an animal, a vertebrate animal, amammal, a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine(e.g., a mouse), canine (e.g., a dog), feline (e.g., a cat), equine(e.g., a horse), a primate, simian (e.g., a monkey or ape), a monkey(e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutan,gibbon), or a human.

In other aspects, the disclosure provides a transgenic mouse whosegenome comprises deletions of the Trpv4 gene in keratinocytes of theepidermis. The transgenic mouse may be a knockout for the Trpv4 gene inkeratinocytes of the epidermis following keratinocyte-specificactivation and expression of a site-specific recombination enzyme.Knockout of the Trpv4 gene may be carried out by any suitable meansknown in the art. For example, the transgenic mouse may be generated byKeratin-14 promoter expression of a site-specific recombination enzyme.Site-specific recombination enzymes may include CRE recombinase. Thesite-specific recombination enzyme may be fused to a mutated estrogenreceptor. An anti-estrogen may have increased affinity to the mutatedestrogen receptor relative to wild-type estrogen. The anti-estrogen maycomprise tamoxifen. In some embodiments, addition of the anti-estrogento the transgenic mouse drives the site-specific recombination enzyme tothe nucleus and results in knockdown of expression of the Trpv4 gene. Assuch, the keratinocyte-specific deletion of the Trpv4 gene may beinduced by applying an anti-estrogen. In some embodiments, deletion ofthe Trpv4 gene can be specifically and conditionally induced inkeratinocytes of the epidermis. In some embodiments, deletion of theTrpv4 gene may be achieved by expression of a constitutively active orinducible recombination enzyme in keratinocytes of the epidermis. Insome embodiments, the transgenic mouse may exhibit reduced expressionrelative to a control of at least one of IL6, ET1, caspase1, IL1β, andCXCL5, or a combination thereof, in response to UVB exposure.

In a further aspect, the disclosure provides methods for identifying aselective inhibitor of TRPA1 and/or TRPV4. The methods may include (a)contacting a mouse with a test compound; (b) determining a biologicalactivity of TRPA1 and/or TRPV4 after contacting with the test compound;and (c) determining a control level of biological activity of TRPA1and/or TRPV4 in the absence of the test compound; (d) comparing thebiological activity of TRPA1 and/or TRPV4 from step (b) with thebiological activity of TRPA1 and/or TRPV4 from a model of TRPV4deletion, wherein the model of TRPV4 deletion includes the transgenicmouse as disclosed herein or a pan-null Trpv4−/− mouse; and (e)identifying the test compound as a selective inhibitor of TRPA1 and/orTRPV4 when at least one of (i) the TRPA1 and/or TRPV4 biologicalactivity is lower in the presence of the test compound than the TRPA1and/or TRPV4 biological activity in the absence of the test compound;(ii) the TRPA1 and/or TRPV4 biological activity in the presence of thetest compound is about the same as, or lower than, the TRPA1 and/orTRPV4 biological activity in the model of TRPV4 deletion; or (iii) anycombination of (i) and (ii).

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including but not limited to”) unless otherwise noted.All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to illustrate aspects andembodiments of the disclosure and does not limit the scope of theclaims.

EXAMPLES Example 1 Materials and Methods

Animals.

The Trpv4 genomic locus was engineered so that loxP sites surroundedexon13 which encodes TM5-6. This mutation was propagated in mice whichwere crossed to K14-CRE-ERtam mice, so that((Trpv4lox/lox)X(K14-CRE-ERtam))-mice could be induced by tamoxifenadministration via oral gavage for 5 consecutive days at 6 mg/day in 0.3mL cornoil, at age 2-4 months of age, plus a one-time booster two weeksafter the last application. Male and female mice were induced equally.Efficiency of knockdown was verified by qRT-PCR for Trpv4 using primerssense 5′-CCTGCTGGTCACCTACATCA (SEQ ID NO: 1) and antisense5′-CTCAGGAACACAGGGAAGGA (SEQ ID NO: 2), with the former primer locatedin exon 13. All animal experimentation described here was conducted infull compliance with NIH and Duke University internal guidelines, andunder a valid IACUC protocol.

Using the same genomic clone that was used for generating the Trpv4−/−pan-null mouse, the Trpv4 targeting construct was electroporated intomouse ES cells (C57BL6 background), and orthotopic integration wasverified by PCR and Southern blot. The engineered mutation wasintroduced into the germline by mating of chimeric mice with C57BL6 WTmice. The selection marker was deleted by FLPemediated excision of thefrt-pGK-neo-frt cassette in FLPe deleter mice. Genotyping wasaccomplished by PCR and subsequent PacI digest (FIG. 1B).

Behavioral Assessment of Withdrawal Thresholds.

Behavioral tests were performed to evaluate the decrease in withdrawalthresholds in response to mechanical von Frey hair or thermal stimuliapplied to hind paws. Tests were conducted. These withdrawal thresholdswere ascertained before and after UV exposure. Mice were exposed using aBio Rad Gel Doc 2000 UV transilluminator (302 nm) for 5 minutes with anexposure of 600 mJ/cm², 3-5 days after the last application oftamoxifen/oil.

Paw Interstitial Fluid Analysis.

48 hours after UV exposure, each animal received an intraplantarinjection of 10 μL PBS directly posteuthanasia. The interstitial fluidwas immediately collected and analyzed by ELISA (Biorad) for presence ofIL-1β.

In-Vivo Topical Interventions.

ET1 injections: After determining base-line withdrawal thresholds, eachanimal received an intraplantar injection of 10 μL 100 nM ET-1 pluscontralateral vehicle. Thresholds were again evaluated 1 hour afterinjection.

GSK205 Topical Treatment:

A viscous solution of 68% EtOH/5% glycerol plus 1 mM or 5 mM GSK205(none for control) was applied to hindpaws by rubbing in 20 μL, appliedat time-points 1 hour and again 10 min before UV exposure.

Formalin-Induced Nocifensive Behavior:

4% formalin was injected into the right hindpaw. Mice were thenvideotaped for 50 mins and behavior analyzed by blinded observers.

Mouse Tissue Processing for 1 μm Semithin Sections and EM.

Samples were processed and subjected to EM.

Mouse and Human Tissue Processing for Immunohistochemistry.

Routine procedures were followed, and human tissue was processed underinstitutional review-board approval (UCSF).

Primary Mouse Keratinocyte Cell Culture.

Primary mouse keratinocytes were derived from back skin of newborn mice.

Analysis of IL-1β Secreted by Cultured Keratinocytes after UV Exposure.

Before UV irradiation, culture media was replaced with PBS. The cellswere then irradiated at a dose of 50 mJ/cm² with UVB. 24 hours later,supernatants were assayed using IL-1β ELISA (R&D Systems, Minneapolis,Minn.).

Ca++ Imaging of Cultured Keratinocytes.

Ca++ imaging of 1° MK was conducted following routine procedures. ForUVB stimulation, a customized device was built. The system comprised aprinted circuit board for electrical interconnects and mechanicalsupport and an ultraviolet light-emitting diode (UV LED). Customizedprovisions at the cellular end included a quartz coverslip as the bottomof the cell culture dish plus a thermal equilibration stage (HW-101Dagan Corporation), fitted to an Olympus BX61 upright microscope. The UVLED was a III-nitride-based type (UVTOP-295 BL; Sensor ElectronicTechnology).

The operating wavelength was 295 nm (FIG. 2A), with a full-widthhalf-max of 12 nm. The optical power was 500 mW. The focal point wasaimed at the plane of the upper surface of the quartz coverslip, whichwas used to minimize UV absorption along the optical path towards thecells (FIG. 2A-C). The optical intensity at the focal point wasestimated to be 150 mW/mm².

Keratinocyte UV Irradiation Using 295 nm LED and Immunocytochemistry.

1° MK were exposed to UVB using the UV optical system (295 nm LED). 24hours later the cells were fixed and fluorescently immunolabeled forET1. Digital images were captured and subjected to morphometry.

Statistical Analysis.

Numeric signals or values were averaged for their respective groups andthe statistical mean+/− standard error of the mean were compared betweengroups by using a fixed-effect one-way ANOVA and post-hoc Scheffe testor Student's t-test, at a significance level of p<0.05.

Chemicals/Biological.

The following biologicals and compounds were used: Endothelin1; BQ123and BQ788 (ET(R) blockers for ET(R)-A and ET(R)-B; Sigma, St. Louis,Mo.), U73122 (PLC inhibitor; Tocris, Ellisville, Mo.), 4α-phorbol 12,13didecanoate (4α-PDD; TRPV4 activator; Tocris), GSK205 (TRPV4 inhibitor(Li et al., 2011; Phan et al., 2009; Vincent and Duncton, 2011)),RN-1734 (TRPV4 inhibitor; Tocris), CGS35066 (endothelin-convertingenzyme inhibitor, Tocris), isopentenyl pyrophosphate, IPP (TRPV3inhibitor; Sigma); and Camphor (TRPV3 activator; Whole Foods).

Behavioral Assessment of Withdrawal Thresholds and Nocifensive Behavior.

Behavioral tests were performed to evaluate the decrease in withdrawalthresholds in response to mechanical or thermal stimuli applied to hindpaws. These withdrawal thresholds were ascertained before and after UVBexposure. Mice were confined by plexi-glass enclosures on top of 25×26cm Bio Rad Gel Doc 2000 UV transilluminator (302 nm), and otherwiseallowed to openly explore this environment. UV-exposure lasted for 5minutes with an exposure of 600 mJ/cm². Careful observations uponinitiation of this method demonstrated that hindpaws were exposed to UVthroughout this period.

Automated Von Frey Hair Testing.

Hindpaw mechanical withdrawal thresholds were determined by theautomated von Frey hair method, using a 0.5 mm diameter stainless steelfilament, part of an automated plantar touch stimulator (Ugo Basile,Modena, Italy). Relevant detail included pre-test acclimatization in aquiet room for 30 min, conducting the test at the same time of day andblinded observers. The stimulus was delivered to the hindpaw,automatically discontinued upon withdrawal, and its intensity recordedautomatically. 6-8 trials per animal were conducted, with equal exposureof both hindpaws, leading to an average withdrawal threshold. Resultsare reported as Δ-threshold, which was calculated by subtractingpost-treatment from pre-treatment measurements, expressed as % orrelative to 1.0.

Hargreaves' Test.

Hindpaw thermal (hot) withdrawal thresholds were determined by thewell-established Hargreaves' method, using an infra-red thermalstimulation device that delivers the stimulus from underneath thehindpaw combined with automatic shut-off upon withdrawal (Ugo Basile).Stimulation and measurements were conducted as described for von Freyhair testing. A cutoff of 20 sec was set to prevent tissue damage.

Formalin-Induced Nocifensive Behavior.

Videos were read by blinded observers for the total amount of time eachmouse spent flinching or licking the injected hindpaw.

Mouse Tissue Processing for 1 μm Semithin Sections and ElectronMicroscopy.

Samples were fixed in 2% glutaraldehyde, 4% PFA, and 2 mM CaCl₂ in 0.05M sodium cacodylate buffer, pH 7.2, at room temperature for >1 h,dehydrated, postfixed in 1% osmium tetroxide, and processed for Eponembedding. Semi-thin sections (1 μm) were stained with toluidine blueand photographed with an Axioplan 2 microscope (Zeiss). For EM analysis,ultrathin sections (60-70 nm) were counterstained with uranyl acetateand lead citrate. EM images were taken with a transmission electronmicroscope (Tecnai G2-12; FEI) equipped with a digital camera (modelXR60; Advanced Microscopy Techniques, Corp.).

Mouse Tissue Processing for Immunohistochemistry.

Routine procedures were followed as described previously (Chen et al.,2009). Mice were perfused transcardially with 30 mL PBS, followed by 30mL 4% paraformaldehyde. Tissues, including the L5 DRGs (bilateral), andfootpad preparations, were dissected and post-fixed in 4%paraformaldehyde. Tissue blocks were further cryoprotected in 30%sucrose in PBS for 24-48 hours. For mouse TRPV4, keratin-specificantibodies, phospho-ERK, IL-6, IL-1β, CXCL5 and caspase-1, tissue wasprepared as frozen blocks and subsequently sectioned on a cryostat. ForCD68, CD15 (neutrophil elastase) and CD3, mouse skin was prepared by 2%PFA perfusion. Footpad and DRG sections (both at 6-10 μm) werethaw-mounted, blocked with 5% normal goat serum (NGS; Jackson), thenincubated overnight at 4° C. with the following primary antibodies:rabbit anti-TRPV4 (1:300; Abcam), mouse anti-keratin 14 (1:300; Abcamagainst C-terminal peptide beyond residue 850); rabbit anti-keratin 14(1:1000; Fuchs-Lab), rabbit anti-phosph-ERK1/2 (1:500; Cell signalingtechnologies), goat anti-IL-1β (1:800; Abcam), goat anti-IL-6 (1:200;Santa Cruz Biotechnology Inc); rabbit anti-caspase-1 (1:200; BiovisionResearch Products, CA); goat anti-mouse LIX/CXCL5 (1:200; R&D SystemsInc), anti-CD68, anti-CD25, and anti-CD3 (AbDSerotec). Immunodetectionwas accomplished with appropriate fluorescently-labeled secondaryantibodies (AlexaFluor595, AlexaFluor488-conjugated antibodies at 1:600;Invitrogen; for CD15 biotinylated secondary antibody from donkey, 1:400followed by rhodamine-streptavidine 1:250), or with peroxidase-linkeddetection reagents (for CD68) for 2 hours at room-temperature. Sectionswere rinsed, mounted, and cover-slipped with fluoromount (Sigma).Digital micrographs were obtained using a BX61 Olympus uprightmicroscope, also with a Zeiss LSM510 confocal, both equipped withhigh-res CCD camera, and acquired with constant exposure settings, usingISEE or Zeiss Zen software. Morphometric analysis was conducted usingImageJ freeware (v1.45) with tailored regions-of-interest that sparedthe nuclear compartment. ImageJ was also used for determination of DRGsurface area.

Human Tissue Specimens Immunolabeling.

Human tissue was deparaffinized with xylene and ethanol series, thenwashed in PBS, and incubated at 80° C. for 20 min in Antigen Retrievalbuffer (Biogenex). Subsequently, specimens were washed in PBS.Endogenous peroxidase was blocked with 0.3% H₂O₂+0.01% sodium azide inPBS for 10 min at room temperature, followed by washing steps in PBS.Blocking was performed in 5% normal horse serum+0.3% Triton-X-100 in PBSfor 1 hour at room temperature. Primary antibodies (anti-TRPV4, Abcam,same as for mouse, 1:8,000; anti-ET1; anti-IL-1b as for mouse tissue)were incubated overnight at 4° C. in Ventana Antibody dilution buffer(Fisher). After washing in PBS, specimens were incubated withbiotinylated donkey-anti-rabbit secondary (Vector, BA-1000), in dilutedblocking buffer for 30 min. After washing with PBS, Avidin Biotin blockwas applied (Vector, PK4000) for 30 min at room temperature, and thepositive immunoreactivity was visualized with DAB (Fisher, NC 9567138).After washing in water, hematoxylin was used to counterstain nuclei.Tissues were washed, dehydrated, and then mounted in Permount (Fisher).For morphometric quantification of TRPV4, IL-1β, and ET1, five sectionsfrom each patient and healthy volunteers (n=3/group) were examined at amagnification of ×20 and photographed. The entire section wasdigitalized using Leica software, and analyzed using ImageJ. Forquantification, DAB and HE staining in 3 randomly selected epidermalregions (3.5×1.25 inches) of each image were separated using the IsoDatathresholding method in the Color Threshold Plugin. Relative signalintensities were calculated from background-corrected measurements.Values are expressed as mean of averages determined from five sectionsper patient. Quantification of human skin tissue for TRPV4, ET1, andIL-1β was performed from acute photodermatitis as compared to healthyskin (n=3 per group). Quantification of various subforms of chronicphotodermatitis as compared to acute photodermatitis and healthy skin iscurrently under active study. More final results await availability andproper staining of at least 3 cases per subgroup of human chronicphotodermatitis.

Primary Mouse Keratinocyte Cell Culture.

The epidermis was separated from the dermis by a 1-hour dispase (BDBiosciences) treatment. Then the keratinocytes were dissociated from theepidermis using trypsin. Keratinocytes were plated on collagen coateddishes or glass or quartz coverslips and grown in keratinocyte serumfree media (Gibco) supplemented with bovine pituitary extract andepidermal growth factor (EGF) (R&D Systems), 100 pmol cholera toxin(Calbiochem, San Diego, Calif., USA) and 1× antibiotics/antimycotics(Gibco), in an incubator at 5% CO₂ and 37° C.

UVB-Stimulation of Cultured Keratinocytes; Calcium Imaging.

Ca++ imaging of mouse 1° MK in response to chemical activation of TRPV4was conducted after loading with 2 μM fura2-AM, following a ratiometricCa2+ imaging protocol with 340/380 nm blue light for dual excitation.Ratios of emissions were acquired at 0.5 Hz. ΔR/R0 was determined as thefraction of the increase of a given ratio over baseline ratio, dividedby baseline ratio. For stimulation of cells with UVB, where fura-2 wasnot suitable because of the proximity of stimulation with 340/380 nm and295 nm, 2 μM fluo4-AM was used instead. Ca++ imaging was carried out at488 nm excitation, acquisition of emissions at 0.5 Hz, expressed asΔF/F0. In the custom-built UV optical system, UV LEDs were capped with aball lens, a transparent optical window in the shape of a hemisphericallens (FIG. 2B). The LED output optical beam focused at 15-20 mm from thelens, with a spot diameter of approximately 1.5˜2.0 mm (FIG. 2C). Theelectrical power supply for the UV LEDs was a surface mount component onthe printed circuit board, which had a steady state 20 mA current outputthat was controlled by an external switch. The thermal equilibrationstage was set for physiological temperature. We confirmed thenon-thermal nature of UVB stimulation using the customized 295 nm LEDdevice in a dedicated experiment (FIG. 2D), thus confirming the specificmodality of stimulation as UVB.

Keratinocyte UV Irradiation Using 295 nm LED and Immunocytochemistry.

Mouse keratinocytes were cultured on collagen coated quartz coverslipsand then stimulated from the bottom using the previously mentioned UVoptical system using the 295 nm LED. 24 hours later the cells were fixedin 4% formaldehyde in PBS for 20 minutes, permeabilized with 0.1% TritonX-100 in PBS for 10 minutes, washed, then blocked in 10% normal goatserum in PBS for 45 minutes. Coverslips were incubated overnight withprimary antibody mouse anti-ET1 (1:200; Abcam), washed three times inPBS and incubated with secondary antibody for 2 hours at 25° C.Coverslips were washed three times in PBS, once with double-distilledH₂O. Digital images were captured using a 40× immersion lens on the BX61Olympus upright microscope. Morphometric analysis was conducted usingImageJ freeware with tailored regions-of-interest.

Determining UVB Permeation of the Skin.

First, the spot size of the UV input optical beam from a LED (UVTOP-295UV) was estimated, as shown in FIG. 16A, using the razor-edge opticalspot occlusion method (results shown in FIG. 16B). The UV LED waspowered with 20 mA of current, resulting in 500 μW of optical power in acircular focal spot 1.5 mm in diameter, with 70% of the total flux in a0.5 mm beam radius. The UV optical power transmitted through the samplewas detected by a Hamamatsu S127-66BR UV detector, and the output of thephotodetector was measured using a Keithley 236 source measure unit.Next, the foot-pad epidermis of a mouse was measured for UV transmissionby placing it on a quartz coverslip and exposure to the UV beam. TheGSK205 was administered to the foot-pad skin in an alcohol and glycerolsolution as for the experiments shown in FIG. 12A. The vehicle-controlgroup was treated with the alcohol and glycerol solution only. Anothercontrol group consisted of a commercially-available SPF100 preparationsunscreen in form of a cream, which was applied similar to thevehicle-control. The data for the GSK205 and sunscreen was normalized tothis control data.

Western Blotting.

Samples were separated by SDS-PAGE, and transferred to PVDF membranes(Bio-Rad). Membranes were blocked with dry milk, then probed withprimary antibodies (rabbit anti-TRPV4 (immunogen=final C-terminalepitope of TRPV4 as for immunolabeling), Alomone; anti-caspase-1,Biovision; mouse anti-β-actin, (clone AC-5) Abcam; mouse anti-β-tubulin,Iowa Hybridoma bank), followed by horseradishperoxidase-conjugatedsecondary antibodies (Jackson Immunoresearch). Secondary antibodies weredetected using Supersignal West Dura Extended Duration substrate(Amersham).

Example 2 Generation of an Epidermal-Specific, Tamoxifen-Inducible Trpv4Null Mouse

To circumvent developmental issues that can arise in gene-targeted micewith ubiquitous deletions, we developed an inducible conditional systemto assess the roles of TRPV4 in UVBmediated skin irritation,inflammation, and sensory sensitization. Using mouse ES cells, we firstbuilt Trpv4lox/lox mice so that the sizable exon coding fortransmembrane domains 5, 6, and the interjacent pore loop was flanked byloxP elements. After crossing to FLPe mice to remove the selectionmarker, flanked by frt elements, these animals were mated with tamoxifen(tam)-inducible, Keratin-14 (K14)-CRE^(ER) transgenic mice. Theconstructs and genotyping are summarized in FIG. 1A-B.

We focused on adult (2 month) glandular mouse paw-pad skin for ouranalyses, as it more closely resembles human skin. Tamoxifen-inductionresulted in efficient knockdown of Trpv4 expression in skin epidermis,as judged by anti-TRPV4 immunolabeling, qRT-PCR and Western blotting(FIG. 3A). However, the gross and microscopic appearance of theskin/epidermis in tam-treated inducible Trpv4 knockout (iKO) mice wasnormal. Given the established dependence of skin renewal on keratinocyteCa++ signaling, we noted that, interestingly, Trpv4-knockdown resultedin no gross alterations in the skin/epidermis nor in the induction ofthe terminal differentiation-specific marker keratin-1 (K1), which isknown to be governed by elevated Ca++ influx suprabasally (FIG. 3A andFIG. 1C). Closer analysis of Trpv4-deficient skin revealed thatexpression of K14 was sustained suprabasally. This keratin is normallydown-regulated at the basal-to-suprabasal transition, concomitant withthe rise in Ca++ signaling and induction of terminal differentiation.

Taken together, despite these more moderate abnormalities, inducingTrpv4 knockdown in keratinocytes at age 8 weeks does not lead to grossinterference with cyto- and layer architecture of the epidermis.

Example 3 Nocifensive Behavior Elicited by UVB Exposure is Dependent onEpidermally-Expressed TRPV4

Underscoring the specificity of Trpv4 gene targeting, peripheral sensoryneurons innervating the footpad still showed robust expression of TRPV4(FIG. 1D). This enabled us to evaluate whether epidermalTrpv4-deficiency critically affects UVB-mediated nocifensive behaviors.For this purpose, we assayed two relevant submodalities—thermal andmechanical stimulation—and compared our iKO mice to pan-null Trpv4−/−and their wild-type (WT) controls (FIG. 3B). 48 hours after UV-exposure,both tamtreated iKO and Trpv4−/− mice displayed much lower sensitivityto noxious radiant heat (Hargreaves' test), also towards noxiousmechanical stimulation (using automated von Frey hair testing), thantheir respective controls. We concluded that epidermal-specific TRPV4deficiency is equivalent to global Trpv4 ablation in reducingUVB-induced behavioral sensitization to radiant heat and mechanicalstimuli, that is, in attenuating thermal and mechanical allodynia.

Further underscoring the importance of epidermal TRPV4 in regulatingnocifensive behavior, a good correlation existed between UV-sensitivityto thermal stimuli and the level of Trpv4 gene knockdown, particularlyat <0.45 the WT levels of Trpv4 mRNA (FIG. 3C). This indicated presenceof a threshold for Trpv4 knockdown to influence nocifensive behavior.Although the dose-responsiveness was less obvious in our mechanicalassay (not shown), we attributed this to the involvement of forced hindlimb (foot) movement in the assay, which will confound the stimulus. Bycontrast, the Hargreaves' assay applies a purely thermal cue whichbecomes noxious without involving confounding stimuli.

In order to assess the specificity of the injurious stimulus, we inducedirritation with foot-pad injections of formalin, eliciting thewell-established bi-phasic response. In this assay, conditionalepidermal knockdown of TRPV4 had no effect on direct peripheral chemicalirritation (phase I) or the early maladaptive neural response (phase II)(FIG. 3D). Taken together, these results suggested that the level ofepidermal Trpv4 knockdown is the determining factor for the degree ofattenuation of nocifensive behavior caused by UVB-irradiation. This isspecific because chemical irritant-induced nocifensive behavior is notaffected by epidermal Trpv4 knockdown.

In additional control experiments, Trpv4lox/+ heterozygous mice hadvirtually identical behavioral sensitization (similar to WT) in responseto UVB, irrespective of CRE-induction with tamoxifen or vehicle (FIG.1E). These findings exclude a functional role for CRE^(ER) on its ownand reiterate the specificity of our approach in targeting Trpv4ablation to keratinocytes. Also, Trpv4−/− skin was equally permeable toUVB as its WT counterpart.

Example 4 Activation Markers of Skin-innervating Peripheral NeuronsSupport Behavioral Findings

In WT mice, the footpad is innervated by sensory neurons of the L5 DRG,which we examined by immunolabeling. TRPV4 expression was unchanged withfoot-pad exposure to UVB, an irritant cue known to sensitize innervatingneurons (FIG. 1D). Interestingly, while sensitization could be verifiedin control mice, it appeared to be absent in tam-treated iKO mice, asdocumented by labeling for phosphorylated ERK (pERK), a known marker ofsensory neuron activation in response to inflammation and irritation(FIG. 3E). Furthermore, size-measurements of pERK-expressing L5 DRGneurons revealed them to be small-to-medium size, suggesting theirpossible involvement in relay of noxious stimuli (FIG. 1F). Theseresults are in good agreement with the nocifensive behavior defects seenin our mice, and further underscore a role for epithelial-expressedTRPV4 in governing UVB-induced activation in skin-innervating DRGsensory neurons.

Example 5 UVB-Induced Skin Inflammation Depends Upon EpidermalExpression of TRPV4

To understand how loss of TRPV4 affects UVB-induced skin, we performedlight microscopy and ultrastructural analyses (FIG. 4A and FIG. 5A-C).In response to UVB, robust signs of inflammation appeared within controlskin, as evidenced by intra-epidermal infiltrates of granulocytes. Withthe epidermis, focal blistering occurred, accompanied by extensivevacuolization. In skin of tam-treated iKO mice with incompletetargeting, inflammatory changes were still observed and were perhapsmoderately less severe. In striking contrast, however, in skin areaswhere conditional epidermal ablation of Trpv4 was complete, no signs ofinflammation or blistering were seen. These data demonstratedconvincingly that epidermal TRPV4 is necessary for skin to mount apro-inflammatory response to UVB exposure. Moreover, since theinflammation involved immune cells and the conditional knockout wasspecific to epidermis, the data further highlight the importance ofepidermal-inflammatory cell crosstalk in the response. Specifically,these data imply that in normal skin, the epidermal keratinocytetriggers inflammatory cell recruitment as part of a UVB response, andthat this circuitry is interrupted when epidermal TRPV4 is knocked down.

We next sought to identify the specific TRPV4-dependent epidermalsignals that occur in WT mice exposed to UVB, and the immune cellpopulations that respond. IL-6 was a suitable candidate for theepidermal signal since it is an established marker of skin epidermalactivation during UV dermatitis, and in addition, IL-6 is robustlyalgogenic. Indeed not only was IL-6 immunoreactivity observed in theUVB-exposed epidermis of control mice, but in addition, this robust IL-6upregulation was virtually eliminated in conditionally targeted as wellas pan-null Trpv4 knockout skin (FIG. 4B and FIG. 5D).

Both macrophages and neutrophils are known to contribute to thereduction of pain thresholds via their expression of a host ofproalgesic/algogenic mediators such as TNFα, IL-6, IL-8, proteases, andchemokines. As judged by immunostaining for CD68 (macrophages) and acell type-specific elastase (indicative of activated neutrophils, alsoknown to enhance nociception), UVB-induced infiltration of both of thesecell populations was markedly reduced in the skin of Trvp4-conditionalknockout mice (FIG. 4C-D). By contrast, the mast cell infiltrate wasunaffected (FIG. 5E), underscoring the specificity of macrophage andactivated neutrophil findings; T-cell count was not changed betweengenotypes either. Taken together, these findings showed that TRPV4expression by keratinocytes is critical for their ability to generateIL-6 and attract macrophages and activated neutrophils in response toUVB radiation.

Example 6 The UVB-Induced Ca++ Response in Primary Mouse Keratinocytesto UVB is Critically Dependent on Extracellular Ca++ Influx ThroughTRPV4

To further dissect the underlying mechanisms involved, we built acustomized device for specific and narrow-band UVB stimulation ofprimary mouse epidermal keratinocytes (1° MK) cultured in vitro (FIG.6A-B and FIG. 2A-D). This allowed use of the Ca++ sensitive dye, fluo-4,and assessment of 1° MK's Ca++ dynamics following UVB exposure (FIG.6B). The elicited Ca++ signal was obliterated by a UV-refracting glasscoverslip, underscoring the strict dependence of the calcium response onUVB (FIG. 6C and FIG. 2A).

Next, we asked whether the UVB-mediated Ca++ response is dependent onextracellular Ca++, and recorded affirmative findings by sequentialexposure to first UVB, then Ca++(FIG. 6D). This finding prompted us todirectly query the role of TRPV4 in the UVB-mediated Ca++ response.Indeed, 1° MK from Trpv4−/− mice exhibited a greatly diminished responserelative to their WT counterparts (FIG. 6E). Moreover, when a selectivesmall molecule-compound, GSK205 (Vincent and Duncton. Current Topics inMedicinal Chemistry 2011, 11, 2216-2226), was used to block TRPV4channel function, WT 1° MK showed a very similar response to that ofTrpv4−/− 1° MK (FIG. 6F).

In view of the known robust expression of TRPV3 in keratinocytes(Moqrich et al., 2005; Peier et al., 2002), we also addressed TRPV3'srole in UVB-mediated Ca++ increase, but observed no effect with theTRPV3-selective inhibitor, IPP (FIG. 6G). The same dose of IPP waseffective in inhibiting camphor-evoked Ca++ transients (FIG. 2E),validating the negative result.

Together, our experiments indicated that UVB exposure to the epidermiselicits the influx of extracellular Ca++ through TRPV4 and not TRPV3channels. Since both channels were present, the data further suggestedthat TRPV4 channels are selectively activated by UVB light. We obtainedcorroborating findings by chemically activating TRPV4 with GSK101, whichcan directly stimulate TRPV4 in WT 1° MK. The GSK101-mediated responsewas dependent upon external Ca++ and was eliminated by the TRPV4inhibitor GSK205 (FIG. 2F). These findings showed that direct chemicalchannel activation of TRPV4 shares critical properties of UVB-evokedCa++ dynamics.

To assess whether TRPV4 is sufficient for the UVB-evoked Ca++ influx, weintroduced high levels of TRPV4 into HEK293 epithelial cells. TRPV4expression endowed these cells with the ability to generate robust Ca++signaling in response to UVB (FIG. 2G). Moreover, if they werepre-exposed to GSK205, the response was blocked. Thus, heterologousTRPV4 expression is sufficient for UVB radiation to cause a cellularCa++ transient.

Example 7 Elevated Endothelin-1 is a Critical Epidermal Effector of theUVB-TRPV4-Ca++ Response

The UVB-TRPV4-Ca++ response depended on upon phospholipase-C (PLC), asit was virtually eliminated by the specific PLC inhibitor, U73122 (FIG.6H). The reliance of TRPV4 activation upon PLC signaling suggested thatPLC's respective lipid products, such as IP3, might be involved. It alsohinted at possible involvement of G protein-coupled receptor signaling.

Using a candidate approach, we focused on endothelin receptors [ET(R)],which are known to be expressed in skin keratinocytes. ET(R)s wereparticularly good candidates since they functionpro-algesic-/algogenically, and their cognate peptide ligand,endothelin-1 (ET1), is elevated when keratinocytes are exposed to UVB.When our 1° MK were exposed to ET1, they exhibited a significantincrease in their UVB-induced Ca++ signaling (FIG. 7A(i)). This responsewas dependent upon TRPV4, as it was greatly diminished by GSK205.Consistent with this result, ET1 augmented GSK101-evoked Ca++ signaling(FIG. 8A).

We next blocked ET1 secretion by applying the proendothelinconvertase-inhibitor, CGS35066. This inhibitor significantly diminishedUVB-induced Ca++ signaling in 1° MK (FIG. 7A(i)). Since ET1's enhancingeffect on Ca++ signaling was dependent upon TRPV4, and the UVB-Ca++response was significantly sustained by ET1 secretion in vitro, weposited that autocrine/paracrine signaling involving ET1 may function toactivate its cognate receptors, ET-R(A) and ET-R(B). In good agreementwith this notion, ET-R inhibitors markedly attenuated the Ca++ signal inresponse to UVB and ET1 co-exposure. Interestingly, antagonism ofET-R(A) eliminated later phases of the Ca++ response, while leaving theinitial rise unaffected; by contrast, antagonism of ET-R(B) convertedthe UVB-Ca++ response into a more protracted one (FIG. 7A(ii)).Co-application of both inhibitors completely eliminated the UVB-inducedCa++ response by 1° MKs (FIG. 7A(iii)).

Taken together, these findings indicate that UVB-mediated ET1 secretionis a significant contributor to the UVB-TRPV4-Ca++ response. The datafurther suggest that independently from UVB's other effects, ET1-ET(R)co-signaling can amplify a TRPV4-dependent Ca++ response. In support ofthis notion, the UVB-Ca++ response could be recapitulated by omittingUVB-exposure and instead co-treating 1° MKs with ET1 and the selectiveTRPV4 activator 4α-PDD. Moreover, this response was significantlyattenuated by ET(R)-A inhibition, and greatly diminished by ET(R)-Binhibition (FIG. 7B). Selective antagonism of ET(R)s led to attenuationfor both compounds, yet showed a slight difference to the patternobserved with UVB, namely co-dependency for both ET(R)s for UVB, andrecruitment of ET(R)-B more than -A for 4α-PDD. Given the pleiotropiceffects of UVB, these minor differences were not surprising, andoverall, the results provided compelling support for the interdependenceof TRPV4 and ET(R) signaling in the response.

Interestingly, un-stimulated 1° MKs produced appreciable levels of ET1,secretory behavior which was dependent upon TRPV4 and PLC (FIG. 8B). Wetested whether UVB causes ET1 upregulation in a TRPV4-dependent manner.Given the exquisite wave-length dependence of ET1 expression in 1° MKs,we resorted to the UVB-LED device as for Ca++ imaging (FIG. 6A and FIG.2A-D). UVB-exposed 1° MKs showed increased ET1 immunolabeling, which wasdiminished when cells were preincubated with TRPV4-inhibitors, GSK205and RN1734 (FIG. 8C-D). Consistent with its effects on UVB-evoked Ca++transients, PLC-inhibitor U73122 also dampened ET1 expression. Together,these findings suggested a limited feed-forward mechanism that involvesTRPV4-dependent increase of ET1 expression in response to UVB, andautocrine/paracrine signaling via ET(R)s. This leads to TRPV4-dependentCa++ signaling, which in turn amplifies ET1 signaling in aparacrine/autocrine fashion.

To assess the physiological relevance of our findings in vivo, weexposed paw-pads to UVB. An interesting time course of Edn1 mRNAexpression was apparent in paw-pad skin of WT mice where it peaked after120 min and relented at 24 hours, but remained significantly elevated.In contrast, there was no regulated expression of Edn1 in paw-pad skinof Trpv4−/− mice. These findings suggest a more direct regulation ofEdn1 gene-expression by TRPV4 in response to UVB. ET1 was readilydetected in control epidermis but reduced in TRPV4-deficient epidermis(FIG. 7C). TRPV4 was also critical for the facilitatory effect of ET1 onnocifensive behavior, as judged by the finding that the withdrawalthresholds in response to von Frey hair stimulation were significantlylowered in control but not Trpv4−/− mice, both pan- and conditionalnull, in response to subepidermal ET1 injections (FIG. 7D). This resultsuggested that ET1's pro-algesic/algogenic effect is fully dependent onTRPV4 in keratinocytes. Although previous studies had shown that ET1 issufficient to elicit nocifensive behavior, the elimination of ET1'spro-algesic/algogenic effect in our Trpv4-conditional null mice wasunexpected given that both ET(R)s and TRPV4 are expressed by sensoryafferents, which were unaffected in our tam-treated iKO mice.

Example 8 UVB-Induced Activation of Inflammasomes by KeratinocytesDepends Upon TRPV4

Another signaling mechanism linking UVB exposure to inflammation andnociception in keratinocytes is the inflammasome, a large multiproteincomplex that assembles in response to infection and other cellularinjury, and triggers an inflammatory cascade culminating incaspase-activation and production of cytokines IL-1 and IL-18.Previously, its formation was shown to depend upon Ca++ signaling,prompting us to query the dependence of inflammasome activation onTRPV4. Indeed, although caspase-1 was upregulated in UVB-treated controlskin, this was largely eliminated in Trpv4−/− and greatly attenuated intam-treated iKO skin (FIG. 9A-B). Moreover, even though caspase-1cleavage (activation) was readily detected by Western blotting oflysates from WT 1° MK exposed to UVB, caspase-1 expression was lackingin Trpv4-null counterparts, even without UVB (FIG. 9C).

Similarly, upregulation of the pro-algesic inflammasome product IL-1βwas readily detected in response to UVB treatment of control skinepidermis but not TRPV4-deficient epidermis. This was demonstrated notonly by immunolabeling but also by measuring IL-1β levels in paw-padedema interstitial fluid (FIG. 9D-F). Together, these results establisha fundamental importance of TRPV4 in keratinocyte-mediated activation ofinflammasomes, which enhance caspase-1-mediated proteolytic cleavage ofpro-IL-1β to form active IL-1β.

Related to IL-1β secretion by skin in response to UVB, we querieddependence of CXCL5 on Trpv4. CXCL5, whose expression is dependent uponIL1β/IL1R1 signaling, has recently been reported to function in aproalgesic/algogenic manner in keratinocytes in response to UVB inrodents and humans. Consistent with the reliance of inflammasomefunction and IL-1β expression on TRPV4, we found that similarly,UVB-induced proalgesic/algogenic CXCL5 upregulation is also dependentupon keratinocyte-derived TRPV4 (FIG. 9G-H).

Example 9 Clinical Relevance of Epidermally-Derived TRPV4 inTransmitting Nociceptive Responses to UVB Exposure

Interestingly, TRPV4 was significantly increased in the epidermis ofhuman patients with UV photodermatitis (FIG. 10A and FIG. 11). Ascompared to healthy skin controls, a robust increase was also seen forET1 and IL-1β immunostaining in acute photodermatitis (FIG. 10A-B).These findings suggest that TRPV4 is also likely to be involved inUVB-induced photodermatitis, one of the various responses of human skinto damaging UV radiation.

In view of the observed impact of epidermal-specific TRPV4-deficiency onmouse nociception in response to UVB, and because of the unambiguouseffects of selective TRPV4 blockers on 1° MK in vitro, we tested thepossible clinical relevance of our findings. For this purpose, wetopically applied TRPV4 inhibitor GSK205 to WT mouse skin andsubsequently exposed animals to UVB (FIG. 12A). While solvent-treatedcontrol mice displayed a normal thermal hypersensitivity response, micetreated with 1 mM GSK205 showed a ˜24 hour delay in sensitivity, andincreasing the dose to 5 mM GSK205 resulted in a sustained attenuationof thermally-evoked nocifensive behavior. Of note, this treatment alsoresulted in a significantly reduced sensitivity of mice to von Frey hairmechanical stimulation.

To assess specificity of the external-topical treatment, we applied 5 mMGSK205 to Trpv4−/− mice vs. vehicle control (FIG. 12A). Nocifensivebehavior did not show any inter-group differences, yet was significantlydifferent from pre-stimulation thresholds. Based upon these results,topical treatment with 5 mM GSK205 in vivo did not elicit off-targeteffects on other channels or signaling pathways that might measurablyinfluence withdrawal behavior. Although GSK205-mediated antagonism ofmacrophage and neural TRPV4 cannot be excluded, the epidermis is theinitial target of topically applied drugs, and the effects we measuredwith 5 mM GSK205 were consistent with those we observed in Trpv4tam-induced iKO skin.

Histopathology of GSK205-topically-treated skin showed hallmarks ofUVB-photodermatitis in vehicle-treated paws, strikingly contrasting toGSK205-treated animals (5 mM), whose paws showed virtual elimination ofinflammation (FIG. 14A). In view of TRPV4-dependence of ET/Edn1expression in skin, we measured Edn1 mRNA abundance in GSK205 vs.vehicle treated paw-pad skin. We detected an early upregulation invehicle-treated mice at the 2 hour time-point which was stillsignificantly elevated over preexposure animals at 24 hours, resemblingEdn1 regulation and time-course in untreated WT mice (FIG. 14B, compareto FIG. 15). In striking contrast, and in keeping with histopathology,Edn1 mRNA-expression in GSK205 topically-treated skin was foundunchanged.

To validate these finding, we tested UVB absorption in GSK205-treatedpaw-pad skin, and whether GSK205 thus functions as sunscreen. Resultswere negative, as demonstrated by equal UVB permeation of GSK205- vs.vehicle-treated paw-pad skin, yet valid, as demonstrated by significantdecrease of UVB permeation with SPF100 sunscreen (FIG. 14CD and FIG.16AB). These results corroborate that effects of topically appliedGSK205 are caused by TRPV4 antagonism, likely by affecting TRPV4 inepidermal keratinocytes.

We also tested the response of down-stream UVB effector mechanisms toGSK205 treatment in vivo. In mouse skin, IL-1β was upregulated inresponse to UVB with vehicle treatment, yet failed to upregulate with 5mM GSK205 (FIG. 12B-C). This in vivo finding was recapitulated in 1° MKwhose UVB-induced increase in IL-1β secretion was completely eliminatedin the presence of 5 μM GSK205 (FIG. 12D). Comparably significant blockswere observed on CXCL5 and IL-6 upregulation (FIG. 13A-B).

Thus, the UVB-evoked signaling in the epidermis was reduced both whenTRPV4 was antagonized by topical application of specific small moleculeinhibitors, and when Trpv4 was targeted genetically in ourkeratinocyte-specific and inducible Trpv4 conditional null mice. Thissuggests ion channel function of TRPV4 to be the critical factor commonto both experimental approaches. Taken together, these findings renderselective TRPV4 blockers, such as GSK205, excellent candidates fortherapeutic approaches to reduce damaging inflammatory responses causedby UVB exposure in humans.

Example 10 Importance of TRPV4 for the Itch-Response

Behavioral studies show reduced scratching behavior in Trpv4 null micecompared to WT mice. Histamine (10%) was injected intracutaneously intothe cheek of C57b/6 control (WT) or Trpv4 null mice (n=6 per group).Over 30 min, Trpv4 null mice showed a significant reduction ((p<0.01)t-test) as compared to WT mice (FIG. 17). The results demonstrated thatsimilar to TRPV1 and TRPA1, TRPV4 is involved in itch, and that blockersof TRPV4 activation may be beneficial for the treatment of itch.

Example 11 Evidence for the Role of TRPV4 in Itch

It was further examined whether TRPV4 has a role in itch using mice witha selective TRPV4 deletion and Compound 48/80. Compound 48/80 (availablefrom Sigma-Aldrich, St. Louis, Mo.) is a well-established pruritogen andelicits histamine-dependent itch by degranulating mast-cells. Mice wereused in which TRPV4 channels had been selectively deleted in skinkeratinoctyes by gene targeting, and the targeted allele was induced byfeeding of tamoxifen, as detailed in Example 2. Wild-type mice were usedas a control.

Compound 48/80 (100 micrograms in 50 μL) was injected retro-auricularlyinto the mice, and mouse scratch behavior in response thereto wasmonitored. Results are shown in FIG. 18. Compared to control mice,scratch behavior over 30 min was significantly reduced for mice in whichTRPV4 channels had been selectively deleted in skin keratinoctyes.

The results provided additional evidence for the role of TRPV4 in itch,and in particular, dependence of itch on the TRPV4 ion channel expressedin keratinocytes of the skin. The results clearly indicated that TRPV4in skin epithelial cells (keratinocytes), and not sensory neurons orimmune-related or allergy-related cells, is the critical site of TRPV4expression and function in histamine-dependent itch. These findings alsosuggested that topical targeting of TRPV4 channels may be successful incombating itch.

Example 12 Overall Preparation of Compounds

The general scheme for preparation of compounds 16-8, 16-12c, 16-13,16-14, 16-16, 16-18, and 16-8/18hy is below, with the following reagentsand conditions for each step: (i) K₂CO₃, CH₃CN; (ii) Zn, MeOH, 12 M HCl;(iii) 1,1′-Thiocarbonyldiimidazole (iv) 7 M NH₃ in MeOH; (v) EtOH,reflux:

Step (i) General Procedure for the SN2 Displacement of 4-NitrophenethylBromide.

Powdered, oven-dried K₂CO₃ (1.5 eq.) and the amine (1.5 eq.) were addedsequentially to a room temperature solution of the bromide (0.33 M) inanhydrous CH₃CN. The reaction mixture was heated to 80° C. (oil bathtemp) until analysis of the reaction mixture by LCMS indicated completeconsumption of the bromide (˜6-18 hours). The mixture was cooled to roomtemperature and diluted with brine (two volume equivalents). Theresulting emulsion was extracted with EtOAc (2× one volume equivalent).The combined extracts were added to silica gel (mass of silica gel=2×mass of starting bromide) and the mixture was concentrated to drynessunder reduced pressure. Flash column chromatography (RediSepRf SiO₂,100% CH₂Cl₂→5% MeOH in CH₂Cl₂) gave the product as a brown to amber oil.The yield of the intermediates 13a-d (the tertiary amines formed in step(i)) are presented in Table 1.

TABLE 1 Yield of tertiary amines 13a-d formed in step (i) IntermediateNo. R n yield 13a Me 0 17% 13b Me 1 49% 13c Me 2 42% 13d Et 1 15%

Step (ii) General Procedure for the Nitro to Aniline Reduction.

A solution of the nitro compound (0.5 M in MeOH) was cooled in anice-NaCl bath. Zinc dust (4.5 eq.) was added in one portion followed bydrop wise addition of 12 M HCl (4.5 eq.) over 2-3 minutes. After 1 hour,the cooling bath was removed, and the reaction mixture was allowed tostir over night at room temperature. The following morning, the mixturewas cooled in an ice-NaCl bath once again and 30% aqueous NaOH was addeddrop wise until pH 14 (universal indicating pH paper) was reached. Themixture was diluted with CH₂Cl₂ (five volume equivalents) and stirredfor 5 minutes. After this time, insolubles were removed at the vacuum,and the filter cake was washed with CH₂Cl₂ (2×25 mL). The organic phaseof the filtrate was separated, washed with brine (100 mL), and dried(MgSO₄). The drying agent was removed by filtration. Silica gel (˜5 g)was added, and the filtrate was concentrated to dryness under reducedpressure. Flash column chromatography (RediSepRf SiO₂, 100% CH₂Cl₂→5%MeOH in CH₂Cl₂) gave the product as a clear, amber oil. The yield of theintermediates 14a-d (the anilines formed in step (ii)) are presented inTable 2.

TABLE 2 Yield of anilines 14a-d formed in step (ii) Intermediate No. R nyield 14a Me 0 75% 14b Me 1 84% 14c Me 2 97% 14d Et 1 85%

Steps (iii) and (iv) General Procedure for Thiourea Formation.

A solution of the aniline (0.22 M) in anhydrous CH₂Cl₂ was added dropwise over 2-5 minutes to an ice-NaCl bath cooled solution of1,1′-thiocarbonyldiimidazole (2 eq., 0.15 M) in anhydrous CH₂Cl₂. After15 minutes, the cooling bath was removed and the reaction mixture wasstirred at room temperature until analysis by TLC (5% MeOH in CH₂Cl₂)indicated complete consumption of the starting aniline. The mixture wascooled once again in an ice bath and 7 M NH₃ in MeOH (10.5 eq.) wasadded drop wise over 2-5 minutes. The bath was removed and the mixturewas stirred over night at room temperature. Silica gel (mass of silicagel=2× mass of starting aniline) was added and the mixture wasconcentrated to dryness under reduce pressure. Flash columnchromatography (RediSepRf SiO₂, 100% CH₂Cl₂→10% MeOH in CH₂Cl₂) gave thepure thiourea. The yield of the intermediates 15a-d (the thioureasformed in steps (iii)-(iv)) are presented in Table 3.

TABLE 3 Yield of thioureas 15a-d formed in steps (iii)-(iv) IntermediateNo. R n yield 15a Me 0 99% 15b Me 1 96% 15c Me 2 88% 15d Et 1 67%

Step (v) General Procedure for Thiazole Formation.

A mixture of the thiourea (0.1 M) in EtOH and the α-bromoacetophenonederivative (1.1 eq.) was heated to 75° C. (oil bath temperature) untilanalysis by TLC (5% MeOH in CH₂Cl₂) indicated complete consumption ofthe thiourea. Silica gel (mass of silica gel=2× mass of startingthiourea) was added, and the mixture was concentrated to dryness underreduced pressure. Flash column chromatography (RediSepRf SiO₂, 100%CH₂Cl₂→10% MeOH in CH₂Cl₂) gave the pure thiazole hydrobromide. Theyield of the final products 16-8 to 16-8/18hy (the thiazolehydrobromides formed in step (v)) are presented in Table 4.

TABLE 4 Yield of thiazole hydrobromides 16-8 to 16-8/18hy formed in step(v) Compound No. R n aryl yield 16-8 Me 1 phenyl 56% 16-12c Me 23-pyridyl 82% 16-13 Me 1 4-pyridyl 83% 16-14 Me 1 2-pyridyl 94% 16-16 Me0 3-pyridyl 98% 16-18 Et 1 3-pyridyl 31% 16-8/18hy Et 1 phenyl 93%

Example 13 Preparation of Compounds 16-8 and 16-8/18hy

Compound 16-8.

Compound 16-8 was prepared as detailed in Example 11. Briefly, asuspension of the bromide (5.01 g, 21.8 mmol), N-benzyl methylamine (4.2mL, 33 mmol, 1.5 eq.) and K₂CO₃ (4.6 g, 33 mmol, 1.5 eq.) in anhydrousCH₃CN (65 mL) was heated to 80° C. (oil bath temp) for 18 hours, afterwhich time the starting material was nearly complete. The mixture wascooled to room temperature and diluted with brine (120 mL). Theresulting emulsion was extracted with EtOAc (2×60 mL). The combinedextracts were added to silica gel (˜10 g) and the mixture wasconcentrated to dryness under reduced pressure. Flash columnchromatography (RediSepRf SiO₂ (120 g), 100% CH₂Cl₂→5% MeOH in CH₂Cl₂)gave the product as a clear, dark orange oil (2.91 g, 49%). ¹H NMR(CDCl₃, 400 MHz): 8.13 (d, J=8.4 Hz, 2H), 7.32 (d, J=8.4 Hz, 2H),7.30-7.22 (m, 5H), 3.55 (s, 2H), 2.91 (t, J=6.8 Hz, 2H), 2.68 (t, J=6.8Hz, 2H), 2.29 (s, 3H). ESIMS: m/z 271 [(M+H)+].

A solution of the nitro compound (2.8 g, 10.4 mmol) in MeOH (20 mL) wascooled in an ice-NaCl bath. Zinc dust (325 mesh, 3 g, 4.5 eq.) was addedfollowed by drop wise addition of 12 M HCl (3.8 mL, 4.5 eq.) over 2-3minutes. After 1 hour, the cooling bath was removed and the reactionmixture was allowed to stir over night at room temperature. Thefollowing morning, the mixture was cooled in an ice-NaCl bath once againand 30% aqueous NaOH was added drop wise until pH 14 (universalindicating pH paper) was reached. The mixture was diluted with CH₂Cl₂(100 mL) and stirred for 5 minutes. After this time, insolubles wereremoved at the vacuum and the filter cake was washed with CH₂Cl₂ (2×25mL). The organic phase of the filtrate was separated, washed with brine(100 mL) and dried (MgSO₄). The drying agent was removed by filtration.Silica gel (˜5 g) was added and the filtrate was concentrated to drynessunder reduced pressure. Flash column chromatography (RediSepRf SiO₂ (120g), 100% CH₂Cl₂→5% MeOH in CH₂Cl₂) gave the product as a clear, amberoil (2.1 g, 84%). ESIMS: m/z 241 [(M+H)+]. This material was used in thenext step without further analysis or purification.

A solution of the amine (2.1 g, 8.7 mmol) in anhydrous CH₂Cl₂ (40 mL)was added dropwise over 2-5 minutes to an ice-NaCl bath cooled solutionof 1,1′-thiocarbonyldiimidazole (95%, 3.1 g, 17.4 mmol, 2 eq.) inanhydrous CH₂Cl₂ (120 mL). After 15 minutes, the cooling bath wasremoved and the reaction mixture was stirred at room temperature for 1.5hours after which time analysis by TLC (5% MeOH in CH₂Cl₂) indicatedcomplete consumption of the starting aniline. The mixture was cooledonce again in an ice bath and 7 M NH₃ in MeOH (13 mL, 91 mmol, 10.5 eq.)was added dropwise over 2-5 minutes. The bath was removed and themixture was stirred over night at room temperature. Silica gel (˜5 g)was added and the mixture was concentrated to dryness under reducepressure. Flash column chromatography (RediSepRf SiO₂ (120 g), 100%CH₂Cl₂→10% MeOH in CH₂Cl₂) gave the thiourea as an amber oil thatsolidified to a tacky residue upon standing (2.5 g, 96%).

A mixture of the thiourea (2.5 g, 8.3 mmol) and 2-bromoacetophenone (1.8g, 9.1 mmol, 1.1 eq.) in EtOH (80 mL) was heated to 75° C. (oil bathtemperature) for 20 minutes after which time analysis by TLC (5% MeOH inCH₂Cl₂) indicated complete consumption of the thiourea. Silica gel (˜5g) was added and the mixture was concentrated to dryness under reducepressure. Flash column chromatography (RediSepRf SiO₂ (120 g), 100%CH₂Cl₂→10% MeOH in CH₂Cl₂) gave the thiazole hydrobromide as a strawcolored glass (3.73 g, 93%).

Compound 16-8/18hy.

Upon examination of the activity of GSK205 relative to compounds 16-8and 16-18, it seemed that removal of a nitrogen from the pyridyl groupincreased the potency of the TRPV4 antagonist, and addition of an extracarbon to the nitrogen carbon side chain increased the potency of theTRPV4 antagonist. Compound 16-8/18hy was formed and based on thestructures of 16-8 and 16-18. See FIG. 19.

Compound 16-8/18hy was prepared as detailed in Example 11. Briefly, asuspension of the bromide (5.01 g, 21.8 mmol), N-benzyl ethylamine (4.9mL, 33 mmol, 1.5 eq.) and K₂CO₃ (4.6 g, 33 mmol, 1.5 eq.) in anhydrousCH₃CN (65 mL) was heated to 80° C. (oil bath temp) for 18 hours, afterwhich time the starting material was nearly complete. The mixture wascooled to room temperature and diluted with brine (120 mL). Theresulting emulsion was extracted with EtOAc (2×60 mL). The combinedextracts were added to silica gel (˜10 g) and the mixture wasconcentrated to dryness under reduced pressure. Flash columnchromatography (RediSepRf SiO₂ (120 g), 100% CH₂Cl₂→5% MeOH in CH₂Cl₂)gave the product as an orange oil that solidified upon standing at roomtemperature (3.3 g, 53%). ¹H NMR (CDCl₃, 400 MHz): 8.13 (d, J=8.4 Hz,2H), 7.32 (d, J=8.4 Hz, 2H), 7.30-7.22 (m, 5H), 3.55 (s, 2H), 2.91 (t,J=6.8 Hz, 2H), 2.68 (t, J=6.8 Hz, 2H), 2.87 (q, J=6.8 Hz, 2H), 1.20 (t,J=6.8 Hz, 3H). ESIMS: m/z 285 [(M+H)+].

A solution of the nitro compound (3.0 g, 10.4 mmol) in MeOH (20 mL) wascooled in an ice-NaCl bath. Zinc dust (325 mesh, 3 g, 4.5 eq.) was addedfollowed by drop wise addition of 12 M HCl (3.8 mL, 4.5 eq.) over 2-3minutes. After 1 hour, the cooling bath was removed and the reactionmixture was allowed to stir over night at room temperature. Thefollowing morning, the mixture was cooled in an ice-NaCl bath once againand 30% aqueous NaOH was added drop wise until pH 14 (universalindicating pH paper) was reached. The mixture was diluted with CH₂Cl₂(100 mL) and stirred for 5 minutes. After this time, insolubles wereremoved at the vacuum and the filter cake was washed with CH₂Cl₂ (2×25mL). The organic phase of the filtrate was separated, washed with brine(100 mL) and dried (MgSO₄). The drying agent was removed by filtration.Silica gel (˜5 g) was added and the filtrate was concentrated to drynessunder reduced pressure. Flash column chromatography (RediSepRf SiO₂ (120g), 100% CH₂Cl₂→5% MeOH in CH₂Cl₂) gave the product as a clear, amberoil (2.3 g, 87%). ESIMS: m/z 255 [(M+H)+]. This material was used in thenext step without further analysis or purification.

A solution of the amine (0.110 g, 0.43 mmol) in anhydrous CH₂Cl₂ (2 mL)was added dropwise over 2-5 minutes to an ice-salt bath cooled solutionof 1,1′-thiocarbonyldiimidazole (95%, 0.162 g, 0.87 mmol, 2 eq.) inanhydrous CH₂Cl₂ (6 mL). After 15 minutes, the cooling bath was removedand the reaction mixture was stirred at room temperature for 1.5 hoursafter which time analysis by TLC (10% MeOH in CH₂Cl₂) indicated completeconsumption of the starting aniline. The mixture was cooled once againin an ice bath and 7 M NH₃ in MeOH (620 μL, 4.3 mmol, 10 eq.) was addeddropwise over 2-5 minutes. The bath was removed and the mixture wasstirred over night at room temperature. Silica gel (˜1 g) was added andthe mixture was concentrated to dryness under reduce pressure. Flashcolumn chromatography (RediSepRf SiO₂ (40 g), 100% CH₂Cl₂→10% MeOH inCH₂Cl₂) gave the thiourea as an amber oil that solidified upon standing(0.130 g, 97%).

A mixture of the thiourea (159 mg, 0.51 mmol) and 2-bromoacetophenone(0.113 g, 0.56 mmol, 1.1 eq.) in EtOH (5 mL) was heated to 75° C. (oilbath temperature) for 1 hour, after which time analysis by TLC (10% MeOHin CH₂Cl₂) indicated complete consumption of the thiourea. Silica gel(˜1 g) was added and the mixture was concentrated to dryness underreduce pressure. Flash column chromatography (RediSepRf SiO₂ (40 g),100% CH₂Cl₂→10% MeOH in CH₂Cl₂) gave the thiazole hydrobromide as astraw colored glass (0.165 g, 78%).

Example 14 Effect on TRPV4-Mediated Calcium Transport

Compounds (GSK205, 16-12, 16-13, 16-14, 16-18, 16-8, and 16-8/18hy) weretested for their effect on TRPV4-mediated calcium influx in N2a culturedcells with targeted expression of human TRPV4. Ca2+ imaging wasperformed according to Li et al. (Environ. Health Perspect. 2011, 119,784-93) and Moore et al. (Proc. Natl. Acad. Sci. U.S.A. 2013, 110,E3225-E3234). Briefly, Ca2+ imaging of primary mouse epidermalkeratinocytes (1° MK) in response to chemical activation of TRPV4 wasconducted after loading with 2 μM fura2-AM, following a ratiometricCa2+-imaging protocol with 340/380 nm blue light for dual excitation.Ratios of emissions were acquired at 0.5 Hz. ΔR/R0 was determined as thefraction of the increase of a given ratio over baseline ratio, dividedby baseline ratio. For stimulation of cells with UVB, where fura-2 wasnot suitable because of the proximity of stimulation with 340/380 nm vs.295 nm, 2 μM fluo4-AM was used instead. Ca2+ imaging was carried out at488 nm excitation, acquisition of emissions at 0.5 Hz, expressed asΔF/F0. TRPV4 was activated with 10 nM GSK101, a specific activator,which had no effect on RFP-transfected cells. Each of the six compoundswere added to a concentration of 2.5 μM, and its effect was observed.

Results are shown in FIG. 20. The bar diagram to the left shows theeffects of 2.5 μM of the respective inhibitor (pre-incubation for 10min). The ordinate is the peak calcium concentration in the cells(average n>75 cells) in nM. All six compounds were effective ininhibiting TRPV4-mediated calcium influx, with 16-18 and 16-8 being themost potent. 16-8/18hy did not show enhanced inhibition over 16-8 (datanot shown). It was noted that GSK205 had a very low potency at thisconcentration in cultured cells with targeted over-expression.

Example 15 In Vivo Pain Model in Mice

Compounds were tested for their effect in reducing pain using an in vivopain model in mice. For mouse formalin-evoked irritant behaviormeasurements, mice were well-fed, well-rested, and tested at the sametime of day, at the same time-point of their circadian rhythm. They wereallowed to acclimate to a plexiglas chamber for at least 30 min beforetesting, and received 10 μL subcutaneous injection of 4% of formalin(diluted from an aqueous solution of commercial 37% formaldehyde withnormal saline (NS)) through a 30-gauge needle into the right whiskerpad,as further detailed in Luccarini et al. (J. Pain, 2006, 7, 908-914).Normal saline was used as control injection. After injection, mice wereimmediately placed back into the chamber and the rubbing behavior wasrecorded by a private consumer-type video-camera for a 45 minobservation period. The recording time was divided into 9 blocks of 5min, and a nociceptive score was determined per block by measuring thetime that the animals spent rubbing the injected area predominantly withthe ipsilateral fore-paw and rarely with hind-paw. This rubbing behaviorwith fore-paw is evoked by pain, which is distinct from itch behavior.Behavioral analysis was conducted by observers blinded to treatment.

To investigate the effects of the specific compounds GSK205, 16-8, and16-8/18hy on formalin-induced nociceptive behavior, mice received asingle subcutaneous injection of the compounds into the whiskerpad (10μL, dissolved in 4% DMSO) 15 min before formalin injection. Controlanimals received the same volume of NS, 4% DMSO.

Results are shown in FIG. 21. FIG. 21A shows the time-course ofnocifensive behavior in response to whisker-pad injection of formalin toBL6 mice. Note the biphasic response and “clean” controls. FIG. 21Bshows quantitation of FIG. 21A, with n=10 animals per group. Note thesignificant increase in response to formalin injection. FIG. 21C showssimilar quantitation when compounds where pre-injected topically at 0.5mM/10 μL. This concentration had no effect on residual nocifensivebehavior in Trpv4−/− mice. Note the lack of effect of GSK205 at thisconcentration, yet a significant attenuation of nocifensive behavior, inparticular of the centrally caused second phase, in response to 16-8 and16-8/18hy. FIG. 21D shows the time course for the three compounds.GSK205, at this concentration, was not different from control, whereas16-8/18hy and 16-8 significantly attenuated nocifensive behavior. Notethe reduction to control levels with the less lipophilic 16-8 at the45-min time-point, and the reversal to control levels with the morelipophilic 16-8/18hy at this time-point. Also note that none of thecompounds influenced the first phase, which was caused by the directirritation that formalin causes on peripheral whisker-pad nerve endings.

Example 16 UVB-Exposure Evoked Nocifensive Behavior

Compounds were tested for their effect on nocifensive behavior (responseto pain) following UVB overexposure. Behavioral tests were performed toevaluate the decrease in withdrawal thresholds in response to mechanicalvon Frey hair or thermal stimuli applied to hind paws. The Von-Freyapparatus (Ugo Basile) applied a mechanical stimulus with a flexiblesteel wire from underneath the hind paw. The force leading to withdrawalwas determined. For the thermal stimuli test, paws were stimulated withheat from underneath applied by an infrared beam (Hargreave's testapparatus; Ugo Basile), and withdrawal latencies were recorded. Thewithdrawal thresholds were ascertained before and after UV exposure.Mice were exposed to UVB 3-5 days after the last application of tam/oil,using a Bio-Rad Gel Doc 2000 UV transilluminator (302 nm) for 5 min withan exposure of 600 mJ/cm². This represents 5-10 times the minimalerythema-inducing dose, in keeping with the rationale of inducingsunburn and studying sunburn-evoked pain.

Results are shown in FIG. 22, demonstrating the effective topicaltreatment of UVB-overexposure evoked nocifensive behavior by compound16-8. Topically applied 16-8 was especially effective in reducing theresponse to pain following UVB exposure, whereas GSK205 at thisconcentration (0.5 mM in 40 μL), was as effective as vehicle(n=4/group).

Example 17 Effect on TRPA1

Compounds were tested for their effect on TRPA1. N2a permanent cellswere transfected with human TRPA1 cDNA, using a pcDNA3.1 expressionplasmid. They were co-transfected with eGFP-expressing plasmid, or, forcontrol, with eGFP plasmid only.

Vehicle-treated TRPA1-expressing cells showed a robust Ca2+ transiencein response to 60 μM AITC and also to 1 mM mustard oil, which are bothknown electrophilic TRPA1-activators. However, eGFP-expressingcontrol-transfected cells (no TRPA1) did not respond to theTRPA1-activator AITC. Cells were then pre-exposed to 5 μM of compounds16-8 and 16-8/18hy for 10 min.

Results are shown in FIG. 23. Averaged Ca2+ signal is shown from >75cells. The resulting Ca2+ transient evoked by 60 μM AITC was reducedby >80% upon administration of compound 16-8 or 16-8/18hy at 5 μM,indicating appreciable TRPA1-inhibitory effects of both compounds.Therefore, compounds 16-8 and 16/8-18hy showed inhibitory activityagainst human TRPA1. For doses below or equaling 25 μM, GSK205 did notinhibit TRPA1 activity.

Example 18 Effect on TRPV1, TRPV2, and TRPV3

Compounds were tested for their effect on TRPV1, TRPV2, and TRPV3.Methods were similar to those described in Example 15. Briefly, N2acells were transfected with human TRPV1, TRPV2, or TRPV3.eGFP-transfection was used as control. For specific stimulation ofTRPV1, 5 μM capsaicin was used. For stimulation of TRPV2, hypotonicity(260 mosmol/L) was used, based on previous reports of TRPV2 beingosmotically responsive. N2a cells were not responsive to hypotonicity,and neither were control-transfected cells. For TRPV3-expressing cells,camphor (20% of a commercially available stock solution) was used.Camphor by itself did not stimulate eGFP-expressing control N2a cells ornative N2a cells. Stimulation and control protocols were applied as forTRPA1-expressing N2a cells as detailed above in Example 15.

Results are shown in FIG. 24, which shows the Ca2+ signals for specificstimulation of TRPV1, TRPV2, and TRPV3. Control-transfected N2a cellsdid not respond to capsaicin (TRPV1 activation) or camphor (TRPV3activation). For TRPV1, TRPV2, and TRPV3, Ca2+ transience was notaffected by administration of 16-8 or 16-8/18hy. Therefore, compounds16-8 and 16-8/18hy did not affect TRPV1, TRPV2, and TRPV3.

Example 19 Proteomics Methodology

Samples were supplemented with 1% Acid-Labile-Surfactant I (ALS-I) in 50mM ammonium bicarbonate and subjected to dithiothreitol reduction andiodoacetamide cysteine alkylation prior to an overnight trypsindigestion at 37° C. (Promega). Resulting peptides were cleaned using a 1cc Oasis MCX cartridge (Waters) following the manufacturer's recommendedprotocol, lyophilized to dryness and resuspended in 10 uL of 0.2% formicacid/2% acetonitrile. Samples were subjected to nanoscale capillaryLC-MS/MS analysis on a nanoAcquity UPLC system (Waters Corp) coupled toa Thermo QExactive Plus high resolution accurate mass tandem massspectrometer (Thermo) via a nanoelectrospray ionization source. Briefly,the sample was first trapped on a Symmetry C18 300 mm×180 mm trappingcolumn for 3 min 5 μL/min (99.9/0.1 v/v water/acetonitrile 0.1% formicacid), after which the analytical separation was performed on a 1.7 μmAcquity BEH130 C18 75 mm×250 mm column (Waters Corp). Peptides wereseparated on a linear gradient from 5 to 40% acetonitrile with 0.1%formic acid over 90 min at a flow rate of 400 nanoliters/minute (nL/min)with a column temperature of 55° C. Data collection on the QExactivePlus mass spectrometer was performed in a data-dependent acquisition(DDA) mode of acquisition with a r=70,000 (@ m/z 200) full MS scan fromm/z 375-1600 with a target automated gain control (AGC) value of 1e6ions followed by 10 MS/MS scans at r-17,500 (@ m/z 200) at a target AGCvalue of 5e4 ions. A 20 sec dynamic exclusion was employed to increasedepth of coverage. Mass spectra were searched using Mascot at 5 ppmprecursor and 0.02 Da product mass tolerances against a SwissProtdatabase with the human and mouse taxonomies selected. Searched spectrawere loaded into Scaffold (Proteome Software) and annotated at a 1%protein false discovery rate. Procedures were as described in Zhang etal. (Curr. Protoc. Mol. Biol., 2014, 108, 10211-102130) and Domon et al.(Science, 2006, 312, 212-217).

Example 20 Analytical Methodology

Sample Processing.

20 μL plasma or tissue homogenate and 40 μL acetonitrile containingappropriate internal standard (see below) were vigorously mixed in aFast-Prep (Thermo-Savant) agitator at speed #4 for 20 s and centrifugedat 13,000 g for 5 min. In case of brain and fat tissue, an additionaldelipidation step was necessary: 20 μL sample was added to 100 μLwater+300 μL chloroform mixture, agitated in Fast-Prep as describedabove, centrifuged, 250 μL of chloroform layer evaporated to dryness,and reconstituted in 60 μL 0.1% formic acid/acetonitrile, 50/50 mixture.Pooled mouse plasma or homogenate from non-treated mouse organs was usedto prepare calibration samples in the appropriate concentration range.Typical range for plasma was 0.5-10 nM, whereas the range for tissueswas adjusted as appropriate. The calibration samples were analyzed alongthe study samples as a single analytical batch.

Liquid Chromatography Tandem-Mass Spectrometry (LC-MS/MS).

The analysis was performed on Shimadzu 20A series LC system coupled withApplied Biosciences/SCIEX API 4000 QTrap MS/MS. Column: Phenomenex, C184×3 mm, AJ0-4287 at 35° C. Solvent A: 0.1% formic acid in MS-gradewater. Solvent B: MS-grade acetonitrile. Elution gradient at 1 μL/min:0-1 min 30-90% B, 1-1.3 min 90% B, 1.3-1.31 min 90-30% B. Run time: 4min. Injection volume: 30 μL. Autosampler temperature: 4° C. The MS/MSparameters (declustering, collision cell, and quadrupole voltages, gasflow, and ionization chamber voltage and temperature) were optimized byinfusion of 100 ng/mL of GSK-205, compounds 16-8 and 16-19 at 10 μL/min.The MRM transitions (m/z), i.e. parent ion/fragment ion, forquantification of the standard (measured analyte) and appropriateinternal standard were as follows: GSK-205 (std.) 401.2/280.2, 16-16(int. std.) 387.1/280.2; compound 16-8 400.1/279.1, 16-16 (int. std.)387.1/280.2; compound 16-19 (std.) 414.2/278.8, 16-8 (int. std.)400.1/279.1. Procedures were as described in Ye et al. (Cell, 2012, 151,96-110).

Example 21 Keratinocyte Histamine Stimulation and Block of the ResultingCa++ Transient by Use of TRPV4 Blockers

FIG. 25 shows Histamine-mediated Ca++ transients in mammalian epidermalkeratinocytes depend on TRPV4. Mouse primary keratinocytes were preparedas described in Moore et al. (Proc Natl Acad Sci USA, 2013, 110,E3225-3416). Cells were pre-treated, for 10 min with 10 μM GSK205. Then100 μM of each, histamine, 2-Pyridylethylamine (selective activator forH1R), and 4-Methylhistamine (selective activator for H4R; both H1R andH4R known to be expressed in rodent and human keratinocytes), wereapplied, and fura-2 loaded keratinocytes were imaged ratiometrically, asdescribed (Moore et al. Proc Natl Acad Sci USA, 2013, 110, E3225-3416;Li, et al. Environ Health Perspect, 2011, 119, 784-9316). Shown in blue(top curve) is the average signal of ≥75 cells. Note the robust signalfor histamine, and in response to selective activator of H1R, lessprominent for H4R, in keeping with previous findings on expressionlevels of these channels in keratinocytes. The histamine-Ca++ responseand the response to activation of H1R could be eliminated completelywith GSK205. The response to H4R could be significantly attenuated whenblocking with TRPV4.

Example 22 Pruritogenic or Algo-Genic

Periostin has a role in chronic allergic skin inflammation and alsopathologic-fibrotic remodeling of the skin. Periostin was injected intomice's footpad to address the question whether it is pruritogenic oralgo-genic. There were no behavioral responses in terms of nocifensiveor scratching behavior of the injected mice when compared tovehicle-control injected animals within the next 30 min, a reasonabletime-frame when examining effects down-stream of the classic pruritogen,histamine.

Example 23 Concentration of TRPV4 Inhibitors in Skin after TopicalApplication to Skin

Inhibiting TRPV4 can readily be accomplished by topical application ofTRPV4-inhibitory compounds. The data show a concentration several timesthe ED50 after topical application, using analytical chemical detectionby liquid chromatography and by mass-spectrometry (Ye, et al. Cell,2012, 151, 96-110). The TRPV4 inhibitory compound 16-19 at 6.2 μM wasfound in their epidermis/dermis preparation (+/−0.83 SEM). For thisexperiment, 5 C57BL6/j mice were subjected to facial skin exposure byapplying 25 microliter solution to their whisker-pad with a cotton swab.This solution contained 50 μM compound. Animals were euthanized 120minutes after compound application, their whiskerpad skin was carefullyprepared as epidermis/dermis sample, and stored in liquid nitrogen untilanalyzed by LC-MS/MS analytical method. For comparison, intra-peritonealapplication of 10 mg/kg bodyweight, in 250 microL vehicle, led to aconcentration in skin of 2.9 μM (+/−0.3) after 6 h and 1.49 μM (+/−0.33)after 24 h. Likely these values under-represent the epidermal anddirectly sub-epidermal concentration of compound because of modestcontamination of the preparation by dermal and sub-cutaneous tissue,also by superficial epidermal layers which are not vital cells.

It was also tested whether compounds are readily broken down in systemicfluid, for which serum was used. A 3 h and 12 h incubation in serum at37° C. did not change the concentration of a fixed spiked amount ofcompounds 16-8, 16-18, and 16-19.

Example 24 Hepatic and Renal Function after Administration ofTRPV4-Inhibitory Compounds

The hepatic and renal function of mice treated with 10 mg/kg body-weightof all three compounds was tested. Hepatic damage marker ALT andcreatinine in serum, drawn at 2 h, 12 h, and 24 h after compoundexposure did not differ significantly from vehicle-control treated mice.All measured concentrations were within normal range for mice, n=6 miceper group (C57BL6/j).

SEQUENCES (SEQ ID NO: 1) 5′-CCTGCTGGTCACCTACATCA (SEQ ID NO: 2)5′-CTCAGGAACACAGGGAAGGA

The invention claimed is:
 1. A method of reducing periostin release fromkeratinocytes in a subject in need thereof, the method comprisingadministering to the subject an effective amount of a TRPV4 and TRPA1inhibitor, wherein the TRPV4 and TRPA1 inhibitor is according to FormulaI:

wherein A, B, and C are independently selected from aromatic,heteroaromatic, cycloalkenyl, and heterocycloalkenyl groups; D is C₁-C₃alkylene; E is a bond, or C₁-C₂ alkylene; and R is selected fromhydrogen, hydroxyl, amino, alkyl, alkenyl, heteroalkyl, aromatic ring,or heteroaromatic ring, and wherein periostin release from keratinocytesin the subject is inhibited upon administration of the TRPV4 and TRPA1inhibitor.
 2. The method of claim 1, wherein the inhibitor does notinhibit TRPV1, TRPV2, or TRPV3.
 3. The method of claim 1, wherein theTRPV4 and TRPA1 inhibitor is selected from the following:

or any combination thereof.
 4. The method of claim 1, wherein B isphenyl.
 5. The method of claim 1, wherein C is phenyl.
 6. The method ofclaim 1, wherein B is phenyl and C is phenyl.
 7. The method of claim 1,wherein A is selected from phenyl and pyridine.
 8. The method of claim1, wherein R is selected from hydrogen, hydroxyl, amino, alkyl, andalkenyl.
 9. The method of claim 1, wherein R is selected from hydrogenand alkyl.
 10. The method of claim 1, wherein D is C₁ alkylene.
 11. Themethod of claim 1, wherein E is C₁ alkylene.
 12. The method of claim 1,wherein E is C₂ alkylene.
 13. The method of claim 1, whereinadministration of the TRPV4 and TRPA1 inhibitor to the subject therebytreats a fibrotic skin disease in the subject, and wherein the fibroticskin disease comprises at least one disorder or condition selected fromscleroderma, scar formation, keloid formation, atopic dermatitis, andskin fibrosis.
 14. The method of claim 1, wherein administration of theTRPV4 and TRPA1 inhibitor to the subject thereby treats a fibrotic skindisease in the subject, and wherein the fibrotic skin disease comprisesat least one disorder or condition selected from keloid formation,chemical or actinic injury, a pathologic response to skin irradiation(“radio-derm”), psoriasis, chronic atopic dermatitis, chronicUV-dermatitis, scleroderma, conditions characterized by a chronicmaladaptive injury response to an ongoing noxious insult to the skin,and scar formation.
 15. A method of reducing periostin release fromkeratinocytes in a subject in need thereof, the method comprisingadministering to the subject an effective amount of a TRPV4 and TRPA1inhibitor, wherein the TRPV4 and TRPA1 inhibitor is according to thebelow:

and wherein periostin release from keratinocytes in the subject isinhibited upon administration of the TRPV4 and TRPA1 inhibitor.
 16. Themethod of claim 15, wherein the inhibitor does not inhibit TRPV1, TRPV2,or TRPV3.